Vaccine for falciparum malaria

ABSTRACT

The invention provides compositions and methods for preventing or reducing the severity of malaria.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/283,472 filed on Feb. 22, 2019, which is a divisional application ofU.S. application Ser. No. 15/607,203 filed on May 26, 2017 (now, U.S.Pat. No. 10,213,502), which is a continuation application of U.S.application Ser. No. 14/361,573 filed on May 29, 2014 (now, U.S. Pat.No. 9,662,379), which is a national stage application, filed under 35U.S.C. § 371, of International Application No. PCT/US2012/067404 filedon Nov. 30, 2012, which claims priority to U.S. Provisional ApplicationNo. 61/641,445, filed May 2, 2012 and U.S. Provisional Application No.61/566,365, filed Dec. 2, 2011, the contents of each are herebyincorporated by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. AI076353awarded by the National Institutes of Health. The Government has certainrights in the invention.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “021486-607D02US_SequenceListing_ST25.txt”, which was created on Feb. 6, 2019 and is 232 KB insize, are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates generally to the field of malaria vaccines.

BACKGROUND OF THE INVENTION

Malaria is a mosquito-borne infectious disease caused by a parasite. Atleast four species of malaria parasites can infect humans under naturalconditions: Plasmodium falciparum (P. falciparum), P. vivax, P. ovaleand P. malariae. The first two species cause the most infectionsworldwide. P. vivax and P. ovale have dormant liver stage parasites(hypnozoites) that can reactivate (or “relapse”) and cause malariaseveral months or years after the infecting mosquito bite; consequently,these species can be difficult to detect in infected individuals. Severedisease is largely caused by P. falciparum while the disease caused byP. vivax, P. ovale, and P. malariae is generally a milder disease thatis rarely fatal.

In humans, the parasites grow and multiply first in the liver cells andthen in the red blood cells. In the blood, successive broods ofparasites grow inside the red cells and destroy them, releasing daughterparasites (merozoites) that continue the cycle by invading other redcells. The blood stage parasites cause the symptoms of malaria. Whencertain forms of blood stage parasites, gametocytes, are picked up by afemale Anopheles mosquito during a blood meal, they start another,different cycle of growth and multiplication in the mosquito. After10-18 days, the parasites are found as sporozoites in the mosquito'ssalivary glands. When the Anopheles mosquito takes a blood meal fromanother human, the sporozoites are injected with the mosquito's salivaand start another human infection when they parasitize the liver cells.

Infection with malaria parasites can result in a wide variety ofsymptoms, typically including fever and headache, in severe casesprogressing to coma or death. There were an estimated 225 million casesof malaria worldwide in 2009. An estimated 781,000 people died frommalaria in 2009 according to the World Health Organization's 2010 WorldMalaria Report, accounting for 2.23% of deaths worldwide. Ninety percentof malaria-related deaths occur in sub-Saharan Africa, with the majorityof deaths being young children. Plasmodium falciparum, the most severeform of malaria, is responsible for the vast majority of deathsassociated with the disease. Children suffer the greatest morbidity andmortality from malaria, yet this age group has not been targeted at theidentification stage of vaccine development. Of the 100 vaccinecandidates currently under investigation, more than 60% are based ononly four parasite antigens—a fact that has caused considerable concern.New antigen candidates are urgently needed.

SUMMARY OF THE INVENTION

The vaccine of the invention successfully and surprisingly elicits animmune response that blocks the Schizont rupture of RBCs (parasiteegress from RBCs), therefore protecting vaccinated individuals fromsevere malaria. The vaccines elicit a strong antibody response to thevaccine antigen, such as PfSEP1 or PfSEP-1A. Due to the permeability ofparasitized red blood cells (RBCs) at the later stages of schizogony,antibodies gain access into the infected RBCs. Antibodies to the vaccineantigen, e.g., a Schizont Egress Protein (SEP) such as PfSEP-1A (SEQ IDNO:2, and other antigenic fragments of the whole protein PfSEP-1 (SEQ IDNO:3)) decrease parasite replication by at least 10% (e.g., 20, 40, 60%,70% or more) by arresting schizont rupture.

Accordingly, the invention features a vaccine for preventing or reducingthe severity of malaria comprising a composition that leads toinhibition of parasite egress from red blood cells or inhibits parasiteegress. For example, the composition comprises a purified polypeptidecomprising the amino acid sequence of SEQ ID NO:2 or a purified nucleicacid encoding a gene product that comprises the amino acid sequence ofSEQ ID NO:2. The vaccine contains one or more compositions of a class ofproteins that are involved in schizont egress such as PfSEP-1/1A (SEQ IDNO:3, 2, respectively), PbSEP-1/1A (SEQ ID NO:67, 68, respectively),PfCDPK5 (SEQ ID NO:47), SERA5 (SEQ ID NO:70, 72), PfSUB1 (SEQ ID NO:74),or PfPKG (SEQ ID NO:76). An immune response elicited by immunizationwith these vaccine antigens inhibits schizont egress. For example, thecomposition comprises a purified antigen that elicits an anti-PfSEP-1antibody response. Alternatively, a passive immunization approach isused. In the latter case, the composition comprises a purified antibodythat specifically binds to one or more of the vaccine antigens that areinvolved in schizont egress (listed above). For example, the compositioncomprises an anti-PfSEP-1 antibody or antigen binding fragment thereof.Thus, a method for preventing or reducing the severity of malaria iscarried out by administering to a subject a composition that inhibitsparasite egress from red blood cells.

The invention also includes a vaccine for preventing or reducing theseverity of malaria comprising a polypeptide composition, wherein thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 2, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46,66 and 72 (antigenic polypeptides or protein fragments). A vaccine forpreventing or reducing the severity of malaria comprising a polypeptidecomposition comprising whole protein antigens such as proteinscomprising the following amino acid sequences: SEQ ID NO: 3, 8, 11, 15,19, 22, 27, 31, 35, 39, 43, 47, 67, 70, 74, and/or 76.

In a preferred embodiment, the invention features an isolated peptidecomprising a peptide having at least 90%, 95% or 99% identity with thesequence of SEQ ID NO: 2; a peptide encoded by a nucleic acid sequencehaving at least 90%, 95% or 99% identity with the sequence of SEQ ID NO:1, or a fragment thereof in a vaccine composition for treatment orprevention of P. falciparum malaria. Alternatively, the isolated peptideof the present invention can be a peptide of SEQ ID NO: 3, a peptideencoded by a nucleic acid of SEQ ID NO: 4, or a fragment thereof.

The present invention also features an isolated nucleic acid sequencecomprising a nucleic acid sequence having at least 90%, 95% or 99%identity with the sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or anyfragment thereof in a vaccine composition for treatment or prevention ofP. falciparum malaria.

Antigens for use in a malaria vaccine include one or more of thefollowing polypeptides (or fragments thereof) that elicit a clinicallyrelevant decrease in the severity of the disease or that reduce/preventinfection or spread of parasites, reduce or inhibit parasite egress froma red blood cell (RBC), reduce or inhibit gametocyte egress (therebyreducing/inhibiting human→mosquito transmission), elicit aparasite-specific antibody or cellular immune response or nucleotidesencoding such polypeptides/fragments: SEQ ID NO: 2, 3, 6, 7, 10, 11, 14,15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 28, 39, 42, 43, 46, 47, 66,67, 70, 72, 74, and/or 76. For example, the vaccine compositioncomprises polypeptides (or nucleic acids encoding them) comprising thefollowing sequences: SEQ ID NO: 2, 10, 14, 18, 22, 26, 30, 34, 38, 42,46, 47, 66, 67, 70, 72, 74, and/or 76.

Also provided herein is a vector or a host cell expressing one or moreisolated peptides or one or more isolated nucleic acid sequencesdescribed herewith.

Another aspect of the present invention relates to a vaccinecomposition. The vaccine composition contains one or more isolatedpeptides or one or more isolated nucleic acid sequences describedherewith. The peptide vaccine may also contain an adjuvant. Exemplaryadjuvants include aluminum salts, such as aluminum phosphate andaluminum hydroxide. Another exemplary adjuvant is an oil adjuvant suchas the Montanide ISA series, e.g., ISA 50 V2 or ISA 720 VG. The DNAvaccine contains a eukaryotic vector to direct/control expression of theantigen in the subject to be treated.

The vaccine of the present invention provides a new regimen in treatingor preventing P. falciparum malaria in a subject. Accordingly, thepresent invention further provides a method of treating or preventing P.falciparum malaria in a subject in need by administering the vaccine tothe subject. Preferably, the subject is a child under 5 years of age.More preferably, the subject is at least about 6-8 weeks of age. Thevaccine is also suitable for administration to older children or adults.The vaccine can be administered orally, parenterally, intraperitoneally,intravenously, intraarterially, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery by catheter orstent, subcutaneously, intraadiposally, intraarticularly, intrathecally,or in a slow release dosage form. Preferably, the vaccine isadministered intramuscularly. The dosing regimen that can be used in themethods of the invention includes, but is not limited to, daily, threetimes weekly (intermittent), two times weekly, weekly, or every 14 days.Alternatively, dosing regimen includes, but is not limited to, monthlydosing or dosing every 6-8 weeks. The vaccine of the present inventioncan be administered intramuscularly once every two weeks for 1, 2, 3, 4,or more times alone or in combination with 1, 2, 3, 4, or moreadditional vaccines in a subject, preferably a human subject. Oneexemplary additional vaccine contains an inhibitor of parasite liverinvasion, such as RTS,S (Mosquirix). Another exemplary additionalvaccine contains an inhibitor of parasite red blood cell invasion, suchas MSP-1. The vaccine can be made by any known method in the art.

Also provided herein are an antibody that specifically binds to anantigen comprising the isolated peptide of the present invention and amethod of treating P. falciparum malaria in a subject in need of byadministering a therapeutically effective amount of such antibody to thesubject. The P. falciparum malaria can be acute P. falciparum malaria.

Also provided herein is a method of treating P. falciparum malaria in asubject in need of by administering a therapeutically effective amountof an antibody described herewith to the subject. Preferably, theantibody is a purified monoclonal antibody, e.g., one that has beenraised to and is specific for the protein of SEQ ID NO:2. For example,the monoclonal antibody is a humanized antibody. The treatment can beinitiated at an early stage after the appearance of recrudescentparasites. The symptoms of the subject may be mild or absent andparasitemia is low but increasing, for example from range4,000-10,000/ul. Alternative, the subject may have fever <38.5° C.without any other accompanying symptom. The subject can be a child under10 years of age. The subject can also be an elder child or an adult. Inone example, the subject is characterized as suffering from acute P.falciparum malaria but has not responded to treatment with anti-malarialdrugs. In this passive immunity approach, the purified humanizedmonoclonal antibody that binds specifically to the protein of SEQ IDNO:2 is administered to the subject to kill the infective agent and/orinhibit RBC invasion.

The antibody can be administered orally, parenterally,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery by catheter or stent, subcutaneously, intraadiposally,intraarticularly, intrathecally, or in a slow release dosage form.Preferably, the antibody is administered intravenously orintramuscularly. For example, the antibody is administered in 1-2 gramamounts, 1, 2, 3, or 4 times. The dosing regimen that can be used in themethods of the invention includes, but is not limited to, daily, threetimes weekly (intermittent), two times weekly, weekly, or every 14 days.Alternatively, dosing regimen includes, but is not limited to, monthlydosing or dosing every 6-8 weeks. The antibody of the present inventioncan be administered intravenously once, twice or three times alone or incombination with 1, 2, 3, 4, or more additional therapeutic agents in asubject, preferably a human subject. The additional therapeutic agentis, for example, one, two, three, four, or more additional vaccines orantibodies, an antimalarials artemisinin-combination therapy, or animmunotherapy. Any suitable therapeutic treatment for malaria may beadministered. The additional vaccine may comprise an inhibitor ofparasite liver invasion or an inhibitor of parasite RBC invasion. Suchadditional vaccines include, but are not limited to, anti-RBC invasionvaccines (MSP-1), RTS,S (Mosquirix), NYVAC-Pf7, CSP, and [NANP]19-5.1.The antibody of the invention can be administered prior to,concurrently, or after other therapeutic agents.

Amounts effective for this use will depend on, e.g., the antibodycomposition, the manner of administration, the stage and severity of P.falciparum malaria being treated, the weight and general state of healthof the patient, and the judgment of the prescribing physician, butgenerally range for the treatment from about 10 mg/kg (weight of asubject) to 300 mg/kg, preferably 20 mg/kg-200 mg/kg.

The present invention further provides a kit for determining thepresence of antibody to P. falciparum in a sample obtained from asubject. A “sample” is any bodily fluid or tissue sample obtained from asubject, including, but is not limited to, blood, blood serum, urine,and saliva. The kit contains an antigen or an antibody of the presentinvention and optionally one or more reagents for detection.

The kit may also contain a sample collection means, storage means forstoring the collected sample, and for shipment. The kit furthercomprises instructions for use or a CD, or CD-ROM with instructions onhow to collect sample, ship sample, and means to interpret test results.The kit may also contain an instruction for use to diagnose malaria or areceptacle for receiving subject derived bodily fluid or tissue.

The kit may also contain a control sample either positive or negative ora standard and/or an algorithmic device for assessing the results andadditional reagents and components. The kit may further comprise one ormore additional compounds to generate a detectable product.

A “vaccine” is to be understood as meaning a composition for generatingimmunity for the prophylaxis and/or treatment of diseases. Accordingly,vaccines are medicaments which comprise antigens and are intended to beused in humans or animals for generating specific defense and protectivesubstance by vaccination.

A “subject” in the context of the present invention is preferably amammal. The mammal can be a human, non-human primate, mouse, rat, dog,cat, horse, or cow, but are not limited to these examples. A subject canbe male or female. A subject can be a child or an adult. A subject canbe one who has been previously diagnosed or identified as havingmalaria, and optionally has already undergone, or is undergoing, atherapeutic intervention for the malaria. Alternatively, a subject canalso be one who has not been previously diagnosed as having malaria, butwho is at risk of developing such condition, e.g. due to infection ordue to travel within a region in which malaria is prevalent. Forexample, a subject can be one who exhibits one or more symptoms formalaria.

A subject “at risk of developing malaria” in the context of the presentinvention refers to a subject who is living in an area where malaria isprevalent, such as the tropics and subtropics areas, or a subject who istraveling in such an area. Alternatively, a subject at risk ofdeveloping malaria can also refer to a subject who lives with or livesclose by a subject diagnosed or identified as having malaria.

As used herein, an “isolated” or “purified” nucleotide or polypeptide issubstantially free of other nucleotides and polypeptides. Purifiednucleotides and polypeptides are also free of cellular material or otherchemicals when chemically synthesized. Purified compounds are at least60% by weight (dry weight) the compound of interest. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight the compound of interest. Forexample, a purified nucleotides and polypeptides is one that is at least90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desirednucleic acid or polypeptide by weight.

Purity is measured by any appropriate standard method, for example, bycolumn chromatography, thin layer chromatography, or high-performanceliquid chromatography (HPLC) analysis. The nucleotides and polypeptidesare purified and used in a number of products for consumption by humansas well as animals, such as companion animals (dogs, cats) as well aslivestock (bovine, equine, ovine, caprine, or porcine animals, as wellas poultry). A purified or isolated polynucleotide (ribonucleic acid(RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequencesthat flank it in its naturally-occurring state. For example, the DNA isa cDNA. “Purified” also defines a degree of sterility that is safe foradministration to a human subject, e.g., lacking infectious or toxicagents.

Similarly, by “substantially pure” is meant a nucleotide or polypeptidethat has been separated from the components that naturally accompany it.Typically, the nucleotides and polypeptides are substantially pure whenthey are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, freefrom the proteins and naturally-occurring organic molecules with theyare naturally associated.

By the terms “effective amount” and “therapeutically effective amount”of a formulation or formulation component is meant a sufficient amountof the formulation or component to provide the desired effect. Forexample, “an effective amount” of a vaccine is an amount of a compoundrequired to blocking red blood cells (RBCs) rupture, block egress ofparasites from RBCs, block gametocyte egress, or elicit an antibody orcellular immune response to the vaccine antigen(s). Ultimately, theattending physician or veterinarian decides the appropriate amount anddosage regimen.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or formulation to a clinically symptomaticindividual afflicted with an adverse condition, disorder, or disease, soas to effect a reduction in severity and/or frequency of symptoms,eliminate the symptoms and/or their underlying cause, and/or facilitateimprovement or remediation of damage. The terms “preventing” and“prevention” refer to the administration of an agent or composition to aclinically asymptomatic individual who is susceptible or predisposed toa particular adverse condition, disorder, or disease, and thus relatesto the prevention of the occurrence of symptoms and/or their underlyingcause.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps and permits those that do notmaterially affect the basic and the characteristic(s) of the claimedinvention.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are bar graphs showing that anti-PfSEP-1 antibodies generatedby DNA vaccination inhibit parasite growth/invasion by 58¬165% across 3parasite strains in vitro. Ring stage 3D7 (A), W2 (B) and D10 (C)parasites were synchronized three times using sorbitol, plated at0.3-0.4% parasitemia, and cultured to obtain mature trophozoites. Maturetrophozoites were cultured in the presence of anti-PfSEP-1 mouse sera(1:10 dilution). Negative controls included no mouse sera and pre-immunemouse sera (1:10 dilution). Sera was heat inactivated and dialyzed priorto use. Parasites were cultured for 24 hrs and ring stage parasites wereenumerated by microscopic examination. Bars represent the mean of 5independent replicates with each replicate performed in triplicate.Error bars represent SEMs. P<0.009 for comparison between pre and postimmune mouse sera by non-parametric Mann-Whitney U test.

FIGS. 2A-D are photomicrographs showing immunolocalization of PfSEP-1.A) methanol fixed infected RBC were probed with mouse anti-PfSEP-1(green) and rabbit anti-MSP-1 (red) and counterstained with DAPI tolabel parasite nuclei. PfSEP-1 is detected only in schizont infectedRBCs, B) methanol fixed schizont infected RBCs do not label when probedwith pre-immune mouse sera, C) non-permeabilized, non-fixed schizontinfected RBCs were probed with mouse anti-PfSEP-1 (red) and rabbitanti-glycophorin A (green) and counterstained with DAPI to labelparasite nuclei. PfSEP-1 co-localized with glycophorin A to the surfaceof schizont infected RBCs, D) non-permeabilized, non-fixed schizontinfected RBCs were probed with mouse anti-PfSEP-1 (5 nm gold particles)and rabbit anti-glycophorin A (10 nm gold particles) and counterstainedwith uranyl acetate to enhance membrane contrast. PfSEP-1 localized tothe schizont/parasitophorous vacuole membrane (black arrow), Maurer'sclefts (yellow arrow) and the inner leaflet of the RBC membrane (greyarrow) while glycophorin A was confined to the outer leaflet of the RBCmembrane (white arrow). Similar results were obtained when PfSEP-1 wasdetected with 18 nm gold particles.

FIGS. 3A-C are bar graphs showing that anti-PfSEP-1 antibodies generatedby DNA vaccination inhibit schizont egress across 3 parasite strains invitro. Ring stage 3D7 (A), W2 (B) and D10 (C) parasites weresynchronized three times using sorbitol, plated at 3.5% parasitemia, andcultured to obtain early schizonts. Parasites were incubated in in thepresence of of anti-PfSEP-1 mouse sera (1:10 dilution). Negativecontrols included no mouse sera and pre-immune mouse sera (1:10dilution). Sera was heat inactivated and dialyzed prior to use.Schizonts were enumerated at 12 hrs post-treatment. Bars represent themean of 5 independent replicates with each replicate performed intriplicate. Error bars represent SEMs. P<0.001 for comparison betweenpre and post immune mouse sera by non-parametric Mann-Whitney U test.Schizontemia was 5.3-6.8 fold higher in post versus pre-immune seratreated cultures.

FIG. 4A is a photograph of an electrophoretic gel, FIG. 4 B is a bargraph showing antibody responses of mice vaccinated with rPbSEP-1A, andFIG. 4C is a line graph showing parasite burden. FIGS. 4A-C show thatvaccination with rPbSEP-1A (recombinant SEP-1A antigenic polypeptidefrom P. berghei) protects mice from challenge with the infectious agent,e.g., P. berghei ANKA. A) rPbSEP-1A was expressed and purified frominduced, clarified E. coli soluble lysates. Recombinant proteincontaining fractions were resolved on an 8-15% SDS PAGE-gel and stainedwith Gel-Code Blue. Lane 1) nickel chelate chromatography of soluble E.coli lysate, lane 2) hydrophobic interaction chromatography of lane 1,lane 3) anion exchange chromatography of lane 2. B) Antibody response ofmice vaccinated with rPbSEP-1A. Following vaccination, mice generatedhigh-titer anti-rPbSEP-1A IgG responses. C) Mice vaccinated withrPbSEP-1A had markedly reduced parasitemia (4.5 fold reduction on day 7post challenge, P<0.002) and parasite growth rate compared to controlmice. All control mice were euthanized on day 7 due to high parasitemiaand associated illness.

FIG. 5 is a line graph showing the incidence of severe malaria and deathin children aged 1.5-3.5 yrs of age during intervals with detectable andundetectable anti-PfSEP-1 antibodies (1,688 and 23,806 weeksrespectively). No cases of severe malaria or death occurred duringintervals with detectable anti-PfSEP-1 antibodies. Error bars represent95% CI adjusted for repeated measures.

FIG. 6 is a dot plot showing the relationship between parasitemia andage for all available blood smears (n=34,038). In multivariateregression analysis, both age (P<0.001) and age2 (P<0.001) were relatedto parsitemia. Second degree (age and age2) polynomial regression lineis depicted in red. Vertical axis is truncated at 1000 parasites/200 WBCfor clarity.

FIG. 7 is a diagram showing the location of SNPs in PfSEP-1. Dataobtained from Plasmodb.org represent sequencing of fifteen lab and fieldisolates. No SNPs are reported in the region identified in thedifferential screening (nt 2,431-3,249).

FIG. 8A-B are diagrams and FIG. 8C is a photograph of an electrophoreticgel. These figures show the knockdown and knockout strategy for PfSEP-1.A) targeting vector for knock down strategy designed to disrupt thepromotor region, B) targeting vector for knock out strategy designed todisrupt protein coding region, C) Evaluation of drug resistant parasitesfor gene disruption. PCR amplification of drug selected parasites wascarried out using: lane 1) F1 and R1 primers, lane 2) F2 and R2 primersand, lane 3) F2 and R3 primers. Only F1 and R1 primers amplifiedsuccessfully indicating the presence of episomal, but not integratedvector.

FIG. 9 is a photograph of an electrophoretic gel showing the results ofchromatographic purification of rPfSEP-1A. Recombinant proteincontaining fractions were resolved on an 8-15% SDS PAGE-gel and stainedwith Gel-Code Blue. Lane 1) induced lysate, lane 2) nickel chelatechromatography of lane 1, lane 3) hydrophobic interaction chromatographyof lane 2, lane 4) anion exchange chromatography of lane 3, lane 5)hydroxyapatite chromatography of lane 4, and lane 6) rPfSEP-1Apost-tangential flow filtration, lyophilization and reconstitution.

FIG. 10 is a bar graph showing differential recognition of rPfSEP-1A byIgG antibodies in plasma from resistant versus susceptible individuals.Antigen coated microtiter wells were probed with plasma pooled fromresistant individuals (clear bars, n=11) or susceptible individuals(black bars, n=14, table S1) and bound antibody was detected withalkaline phosphatase conjugated goat anti-mouse IgG. RAMA-E is a P.falciparum merozoite protein, BSA is bovine serum albumin. Barsrepresent mean of 4 replicate wells. Error bars represent SEM.Recognition of rPfSEP-1A by antibodies in resistant plasma, as assessedby optical density, was 4.4 fold higher than by antibodies insusceptible plasma (Student's t-test, P<0.0002).

FIGS. 11A-B are photographs of electrophoretic gels showing that anti-PfSEP-1 antibodies recognize a 244 kDa protein in P. falciparum extracts.Mixed stage 3D7 infected RBCs, uninfected RBCs and rPf SEP-1A wereanalyzed by western blot. A) lanes 1 and 3-3D7 infected RBC extracts,lanes 2 and 4-uninfected RBC extracts. Lanes 1 and 2-probed withanti-PfSEP-1 antisera (1:500), lanes 3 and 4-probed with pre-immunemouse sera (1:500). B) lanes 1 and 2-0.05 ug of rPfSEP-1A, lane 1-probedwith anti-Pf SEP-1 mouse sera (1:2000), lane 2-probed with pre-immunemouse sera (1:2000).

FIGS. 12A-B are bar graphs showing that anti-rPfSEP-1A antibodiesgenerated by protein immunization inhibit parasite growth/invasion by72-74% across 2 parasite strains in vitro. Ring stage 3D7 (A), and W2(B) parasites were synchronized three times using sorbitol, plated at0.3-0.4% parasitemia, and cultured to obtain mature trophozoites. Maturetrophozoites were cultured in the presence of anti-rPfSEP-1A mouse sera(1:10 dilution). Negative controls included no mouse sera and pre-immunemouse sera (1:10 dilution). Sera was heat inactivated and dialyzed priorto use. Parasites were cultured for 24 hrs and ring stage parasites wereenumerated by microscopic examination. Bars represent the mean of 5independent replicates with each replicate performed in triplicate.Error bars represent SEMs. P<0.009 for comparison between pre and postimmune mouse sera by non-parametric Mann-Whitney U test.

FIGS. 13A-B are photomicrographs showing that PfSEP-1 is not detected introphozoite infected RBCs or non-infected RBCs. Non-permeabilized,non-fixed trophozoite infected RBCs (A) or uninfected RBCs (B) wereprobed with mouse anti-PfSEP-1 (5 nm gold particles) and rabbitanti-glycophorin A (10 nm gold particles) and counterstained with uranylacetate to enhance membrane contrast. PfSEP-1 was not detected introphozoite infected RBC or uninfected RBCs, while glycophorin A wasconfined to the outer leaflet of the RBC membrane (white arrow).

FIG. 14A is a bar graph, and FIG. 14B is a photomicrograph showing thatanti-rPfSEP-1A antibodies generated by protein immunization inhibitschizont egress across 2 parasite strains in vitro. A) Ring stage 3D7(top panel), and W2 (bottom panel) parasites were synchronized threetimes using sorbitol, plated at 3.5% parasitemia, and cultured to obtainearly schizonts. Parasites were incubated in in the presence of ofanti-PfSEP-1 mouse sera (1:10 dilution). Negative controls included nomouse sera and pre-immune mouse sera (1:10 dilution). Sera was heatinactivated and dialyzed prior to use. Schizonts were enumerated at 12hrs post-treatment. Bars represent the mean of 5 independent replicateswith each replicate performed in triplicate. Error bars represent SEMs.P<0.009 for comparison between pre and post immune mouse sera bynon-parametric Mann-Whitney U test. Schizontemia was 4.3-6.0 fold higherin post versus pre-immune sera treated cultures. B) Representativemicrographs of giemsa stained blood films prepared from 3D7 culturestreated with pre-immune (top panel) and post-immune (bottom panel) sera.

FIGS. 15A-C are bar graphs. Parasite density on A) all blood smears andB) positive blood smears in children aged 2-3.5 yrs during intervalswith detectable and undetectable anti-PfSEP-1 antibodies, afteradjusting for hemoglobin phenotype, age, average prior parasitemia onall blood smears, and repeated measures. Error bars represent SEM. C)Incidence of mild malaria in children aged 2-3.5 yrs of age duringintervals with detectable and undetectable anti-PfSEP-1 antibodies afteradjusting for hemoglobin phenotype, age, average prior parasitemia onall blood smears, and repeated measures. Error bars represent 95% CI.

FIG. 16 is a table showing epidemiological characteristics of resistantand susceptible individuals used in differential screening assays.

FIG. 17 is a table showing epidemiological characteristics of resistantand susceptible individuals used in confirmatory ELISA assays.

FIGS. 18A-G are photomicrographs showing the results of animmunofluorescence analysis on methanol fixed infected red blood cells(iRBCs) using mouse anti-PfSEP-1 sera.

FIG. 19 is a bar graph showing growth inhibition assay. Rabbitanti-PfSEP-1 inhibits parasite growth/invasion by 68% in vitro.

FIG. 20 is a diagram showing mechanisms of schizont egress andprotein-protein interactions involved in the process.

FIG. 21A-B are diagrams showing intracellular proteins and theirinteractions in uninfected RBCs (A) compared to parasite infected RBCs(B). FIG. 21 B illustrates the role of PfSEP in and protein-proteininteractions involved in schizont egress.

DETAILED DESCRIPTION

The invention represents a significant breakthrough in the treatment orprevention of malaria, for example, such as P. falciparum malaria. Priorto the present invention, an effective vaccine was not yet available formalaria, although several vaccines are under development. The vaccine,SPf66, was tested extensively in endemic areas in the 1990s, butclinical trials showed it to be insufficiently effective. Other vaccinecandidates, targeting the blood-stage of the parasite's life cycle, suchas anti-red blood cell (RBC) invasion (P. falciparum merozoite specificprotein 1 (MSP-1) antigen and P. falciparum merozoites Apical MembraneAntigen 1 (AMA-1) antigen), have also been insufficient on their own.Several potential vaccines, for example, RTS,S (also called Mosquirix)targeting the pre-erythrocytic stage are being developed. One majorchallenge in the field is short acting time for a vaccine due to thequick infection/life cycle of the parasite. A vaccine, such as RTS,S,functioning at pre-liver stage has only 5 minutes to act beforesporazoite enters hepatocytes. Anti-RBC invasion vaccines have only 15seconds before merozoite enters RBCs.

P. falciparum remains a leading cause of morbidity and mortality indeveloping countries and vaccines for this parasite are urgently needed.Human residents of endemic areas develop protective immunity that limitsparasitemia and disease. The subject invention relates to nucleic acidand polypeptide sequences designed from P. falciparum in a vaccinecomposition. The vaccine antigens were identified using a differentialscreening strategy using sera from resistant individuals and fromsusceptible ones. Antigens were identified by binding to antisera fromresistant individuals were further characterized. Such nucleic acidsequences and polypeptides were found to be useful for therapeutic aswell as diagnostic purposes.

Polynucleotide Sequence and Encoded Polypeptides

The invention is directed in part to P. falciparum polynucleotides andpolypeptides that are useful, for example, for antigens for vaccinesagainst P. falciparum malaria.

Human residents of endemic areas develop protective immunity that limitsparasitemia and disease, and naturally acquired human immunity providesan attractive model for vaccine antigen identification. Plasma samplesand parasitologic data collected during a longitudinal birth cohortstudy in Muheza, Tanzania (TZN) were used to identify previously unknownP. falciparum antigens associated with resistance during early life. Theantigens were then validated as targets of antibodies associated withresistance to parasitemia in a large cohort of young children.

Using plasma obtained from maximally resistant and susceptible membersof the Muheza cohort, parasite antigens recognized by host antibodiesthat mediate resistance to parasitemia were identified.

750,000 phage from a 3D7 based blood stage P. falciparum library weredifferentially screened using pooled plasma from the resistant andsusceptible individuals. Three clones that are uniquely recognized byantibodies in the plasma of resistant but not susceptible pools wereidentified. These clones encode MSP-7 (MSP-7 nts 200-1,052), a uniquehypothetical gene on Ch10 (Chromosome #10 bp 901175 to 900359), and aunique hypothetical gene on Ch11 (Chromosome #11 nts. 1333936 to1335849). The gene on Ch11 has the gene ID of PF10_0212a.

Clone #2: Plasmodb.org designation: Gene PF10_0212a (Version 9.2)Nucleic acid sequence of Clone #2, 819 bp (Sequence 2,431-3,249 of gene PF10_0212a)AACGAGGATAGAGGAATATACGATGAATTATTAGAAAATGATATGTGTGATTTATACAATTTAAAAATGCATGATTTGCATAATTTAAAATCCTATGATTTTGGATTATCTAAAGATTTATTAAAAAAGGATATTTTTATATATAGTAATAATTTGAAAAATGATGATATGGATGATGATGATAATAATAATATGAATGATATTGCTATAGGTGAAAATGTAATATATGAAAATGATATACATGAAAATAATATAGATGATAATGATATGTATAATAATTACGTGAATGGAAATGATTTATATATTAACAATATGCAGGATGATGCCATGGACGATATTGTATATGATGAGGAAGAAATTAAAAGCTTCCTAGATAAATTAAAATCTGATATATCAAATCAAATGAATGTAAAAAATGGAAATGTCGAAGTTACAGGAAATGGTGGTAATGAAGAAATGTCTTATATAAATAATGATGAAAATTTACAAGCTTTTGATTTGTTAGATAATTTCCATATGGATGATTATGGTAATAATTATAATGATAATGAAGAAGATGGGGATGGGGATGGGGATGACGATGAACAGAAGAAAAGAAAACAAAAAGAGTTACATAATGTAAATGGAAAATTAAACTTATCAGATTTAAATGAATTAAATGTAGATGATATAAATAATAATTTTTATATGTCAACTCCTCGAAAATCTATAGATGAACGTAAAGATACGGAATGTCAAACAGATTTTCCCTTATTAGATGTATCAAGGAATACTAATAGGACTCCTAGAAGAAAAAGTGTGGAAGTAATACTTGTAGAA (SEQ ID NO: 1) Sequence Length: 819Amino acid sequence of Clone #2 (a.k.a., PfSEP-1A)NEDRGIYDELLENDMCDLYNLKMHDLHNLKSYDFGLSKDLLKKDIFIYSNNLKNDDMDDDDNNNMNDIAIGENVIYENDIHENNIDDNDMYNNYVNGNDLYINNMQDDAMDDIVYDEEEIKSFLDKLKSDISNQMNVKNGNVEVTGNGGNEEMSYINNDENLQAFDLLDNFHMDDYGNNYNDNEEDGDGDGDDDEQKKRKQKELHNVNGKLNLSDLNELNVDDINNNFYMSTPRKSIDERKDTECQTDFPLLDVSRNTNRTPRRKSVEVILVE (SEQ ID NO: 2) Sequence Length: 273Amino acid sequence of PF10_0212a (PfSEP-1)MMENKYPNELFCYINRYNINEIIENGEEKYVNEYDEDKNMSINHMNENDGICEYEIPFLLDYVDDSNKEDSEKNSLKSYLDDGASTILSKPDELENYNKQNENEFDENNNNKNNKIDQLKEKINIIIIPNKGVINNFEEILSMANRNDKNIEKKLNDRFYQICCKSIADINTHNLNKIKDLKKKKNNKGSLNIEHIDYGDIFLTIHDTLKSNNKIKGNNKTNLLHDSSYEIKKKTRRGTNIYKNPFHHRGSYLTSYENQKDIIYLNNLNNIMMDKYSNCSDSRKKEYSHFNSQEFSYDKYSMKDRMFLKNLYMKQNRLRDKRGKYHKLGDYQNIENYRKTGEHSFDCMNMSDIMHSNKMSHVNIMDHMIYKDNNNMSKLVDTINSREKDVKNYDDNFESYNNFFKNNNDEQHICLEYDDTYNLKDTVKNIIVEEEQCGKGVACICDKNEDVDDLFVSKKTNYSSNKKREDYEKVFLEDNLHLKQTPSKRTKINIIPDYYDNNRSNKSYKENEEDALFEVCGSLKNDDILYKDNKLNVINEDNIKEEDDKESVVHLDNDEDKKEEMYKDVYPNVLSCEKETIRRNEKYNKSLNSTSSFEKIDNPSEINVESKEDTEYFDLLIKKYEDTKINVYDNESLLLDLSNELREEMAKGDSNKNVNKVEDNDNKKENICHDNIMEDICHNNNVEDMYRNNNVEDMYRNNNVEDMYRNNNVEDMYRNNNVEDVCHNNNVEDVCHNNNVEDVCHNNNVEDVYHNNNVEDMYHDNNIEDVCHNNNVEDVCHNNNVEDHVNYDNEELNKKMDEMKEEKEERNEDRGIYDELLENDMCDLYNLKMHDLHNLKSYDFGLSKDLLKKDIFIYSNNLKNDDMDDDDNNNMNDIAIGENVIYENDIHENNIDDNDMYNNYVNGNDLYINNMQDDAMDDIVYDEEEIKSFLDKLKSDISNQMNVKNGNVEVTGNGGNEEMSYINNDENLQAFDLLDNFHMDDYGNNYNDNEEDGDGDGDDDEQKKRKQKELHNVNGKLNLSDLNELNVDDINNNFYMSTPRKSIDERKDTECQTDFPLLDVSRNTNRTPRRKSVEVILVEKKLKKKKQKCMDKYTDANEDSNRRYPKRNRIKTLRYWIGERELTERNPYTGEIDVVGFSECKNLQDLSPHIIGPIEYKKIYLKNLNSNEHEENEDNNGDIIENNNGDVIENNNGDIIEDNNANEKNHNNLESEGKGIVYDDVNNLHVHTNSDNSAHSKKIKGAPSRFSNTNNGRKKRRRRKFINVVNYIKKKKKKKLIKSMDNMEVTDNFKNDMSDENKQSGDENKQSGDENKQSGDENKQSGDENKQTNNDIKQSDNDIKQSDDIYMNEDMNLFNDLNDNFDNNEYFINNGDKDSHAEEEMAIENIQSKSIEKDILNNEEQDNNNIFDIDNELIDMKDGNVDEMESDEKLKTFEKLESLKSTTHLNNTDNCDVNLSEQTNEINYDEEKKVNKKTNHEKMKKKKKKKKKKKKKKKKEKKQIDIMYKNLSRLNLNLLLPTKKKVKKSKNSFKKEEEKQKKKNKKVKKIKGINKGEKIKSNKKENKDNNNDSSTECVVEGEKGKDLHEFNKNGNLEDEQMDVDISMNISSINCESDNKNVSKEGEEEKKDIAENKEEVDKNKEEVYMDKHEMDLNNEEVYMDKNEMDLNNEEVYMDKHEMDLNNEEVYMDKHEMDLNNEEVYMDKHEMDLNKEEVYMDKHEMDLNNEEVDKENEYDENILSDNIIYNENNSFGNNKNSFFNNTSPLKTEIINEEENSLNEMKEDINEYVEMENKLDTEKIKDSEKIGGKIEVDNKMISPINRHNFYLTILEGMNKNFPRQWNKNNITLSKNQGQIYKGRKEKKRKRSYRNDEKLLDHSILNDINISDKMDERNELLESIKSNSTINNVLEIIKYDNRKKIKKNDTNKEIIKYDNFTSKYNNKSNDIQLNGGIYINKFKLSLDMPINKLAVSSNLGPPSSIGSTEIQPIQKNFNDFKMNINVYCIRMEPHEKYSSYSHKNNLVVYIDKGEKINIIINMSKTYEKGDFFYIPRFSNFQIINDSRCDCVLYVCPLI (SEQ ID NO: 3) SequenceLength: 2074 aa; underlined sequence corresponds to PfSEP-1Aantigenic fragment. Coding Nucleotide sequence of PF10_0212a (PfSEP-1)ATGATGGAAAATAAATACCCAAATGAATTATTCTGTTATATAAATAGATATAATATAAACGAAATAATAGAAAATGGAGAAGAGAAGTATGTAAATGAATATGATGAAGATAAGAATATGTCAATAAATCATATGAATGAAAACGATGGTATATGTGAATATGAAATACCATTTTTATTAGACTATGTGGATGATAGTAATAAAGAAGATTCAGAGAAAAATTCATTAAAGAGTTATCTCGATGATGGTGCATCCACTATCCTTTCAAAACCAGATGAACTGGAAAATTATAATAAACAAAATGAAAATGAATTTGACGAAAATAATAATAATAAAAATAATAAAATTGACCAATTGAAGGAAAAAATAAATATTATAATAATACCAAATAAAGGTGTTATAAACAATTTTGAAGAGATATTAAGCATGGCAAATCGTAATGATAAAAATATAGAGAAAAAGTTGAATGATAGATTTTATCAAATATGTTGTAAAAGTATAGCTGATATAAACACACACAATTTAAATAAAATTAAAGATTTGAAAAAAAAAAAAAATAATAAAGGATCCTTAAATATTGAACATATAGATTATGGAGATATTTTTCTTACTATACATGATACATTAAAAAGTAATAATAAAATAAAAGGAAACAATAAAACTAACTTATTACACGATTCTTCTTATGAAATAAAAAAGAAAACAAGAAGAGGAACAAATATATATAAAAATCCATTTCATCATAGAGGTTCCTATTTAACTTCGTATGAAAATCAAAAGGATATCATTTACCTTAATAATTTAAACAACATTATGATGGATAAATATAGTAATTGTAGTGATTCACGAAAAAAGGAATATTCGCATTTCAATTCGCAGGAGTTTTCATATGATAAATATAGTATGAAAGACAGAATGTTTCTCAAAAATTTGTATATGAAACAAAATAGATTAAGAGATAAAAGGGGGAAATATCACAAATTGGGAGATTATCAAAATATTGAAAACTATCGTAAAACGGGTGAACATAGTTTTGATTGTATGAATATGTCAGATATTATGCATTCAAATAAAATGAGCCATGTTAATATCATGGATCATATGATATATAAAGATAATAACAATATGAGCAAACTAGTAGATACAATAAATTCTCGTGAAAAGGATGTAAAAAATTATGACGATAACTTTGAAAGCTATAATAATTTTTTTAAGAATAATAATGATGAACAACATATATGTTTGGAGTATGACGATACATATAACTTAAAAGATACAGTTAAAAATATTATTGTTGAAGAAGAACAATGTGGTAAGGGTGTTGCTTGTATATGTGATAAGAACGAAGATGTTGACGATTTGTTTGTTTCAAAGAAAACGAATTATTCTTCTAATAAAAAAAGAGAAGATTATGAGAAAGTATTTCTTGAAGATAATTTACATTTAAAACAAACTCCATCAAAAAGAACAAAAATTAATATAATCCCAGATTATTATGATAACAATAGAAGTAATAAGAGTTATAAGGAAAATGAAGAGGATGCTTTGTTTGAGGTATGTGGTAGTTTAAAAAACGATGATATATTGTATAAAGATAATAAGTTGAATGTCATAAATGAAGATAATATAAAGGAAGAGGATGACAAAGAAAGTGTTGTTCATTTAGATAATGATGAGGATAAAAAAGAAGAAATGTATAAAGATGTATATCCCAATGTATTGTCTTGTGAAAAAGAAACGATTAGGAGGAATGAAAAGTATAACAAATCATTGAACAGTACAAGTAGCTTTGAAAAAATTGATAATCCAAGTGAAATTAATGTTGAAAGTAAGGAAGATACAGAATATTTTGATTTATTAATAAAAAAATATGAGGATACAAAAATAAACGTATATGATAATGAATCTCTTTTATTGGATCTTAGTAATGAGCTACGTGAAGAAATGGCCAAGGGGGATTCTAATAAAAATGTAAATAAAGTGGAAGATAATGATAATAAAAAGGAAAATATTTGTCATGATAATATCATGGAAGATATTTGTCATAATAATAACGTGGAAGATATGTATCGTAATAATAACGTGGAAGATATGTATCGTAATAATAACGTGGAAGATATGTATCGTAATAATAACGTGGAAGATATGTATCGTAATAATAACGTGGAAGATGTTTGTCATAATAATAACGTGGAAGATGTTTGTCATAATAATAACGTGGAAGATGTTTGTCATAATAATAACGTGGAAGATGTTTATCATAATAATAACGTGGAAGATATGTATCATGATAATAACATTGAAGATGTTTGTCATAATAATAACGTGGAAGATGTTTGTCATAATAATAACGTGGAAGACCATGTTAATTATGATAATGAAGAATTGAATAAAAAAATGGATGAGATGAAAGAAGAAAAGGAAGAAAGAAACGAGGATAGAGGAATATACGATGAATTATTAGAAAATGATATGTGTGATTTATACAATTTAAAAATGCATGATTTGCATAATTTAAAATCCTATGATTTTGGATTATCTAAAGATTTATTAAAAAAGGATATTTTTATATATAGTAATAATTTGAAAAATGATGATATGGATGATGATGATAATAATAATATGAATGATATTGCTATAGGTGAAAATGTAATATATGAAAATGATATACATGAAAATAATATAGATGATAATGATATGTATAATAATTACGTGAATGGAAATGATTTATATATTAACAATATGCAGGATGATGCCATGGACGATATTGTATATGATGAGGAAGAAATTAAAAGCTTCCTAGATAAATTAAAATCTGATATATCAAATCAAATGAATGTAAAAAATGGAAATGTCGAAGTTACAGGAAATGGTGGTAATGAAGAAATGTCTTATATAAATAATGATGAAAATTTACAAGCTTTTGATTTGTTAGATAATTTCCATATGGATGATTATGGTAATAATTATAATGATAATGAAGAAGATGGGGATGGGGATGGGGATGACGATGAACAGAAGAAAAGAAAACAAAAAGAGTTACATAATGTAAATGGAAAATTAAACTTATCAGATTTAAATGAATTAAATGTAGATGATATAAATAATAATTTCTATATGTCAACTCCTCGAAAATCTATAGATGAACGTAAAGATACGGAATGTCAAACAGATTTTCCATTATTAGATGTATCAAGGAATACTAATAGGACTCCTAGAAGAAAAAGTGTGGAAGTAATACTTGTAGAAAAAAAATTAAAAAAAAAAAAACAGAAATGTATGGATAAATATACAGATGCAAATGAGGATAGTAATAGAAGATATCCCAAAAGAAATCGAATTAAAACTTTGCGTTATTGGATAGGAGAAAGAGAGTTAACTGAAAGAAACCCTTACACAGGAGAAATAGATGTTGTAGGATTTAGTGAGTGTAAAAATTTGCAAGATTTGTCACCTCATATTATTGGTCCGATTGAATATAAAAAAATATATTTGAAAAATCTTAATAGTAATGAACATGAGGAAAATGAAGATAATAATGGAGACATTATTGAAAATAATAATGGGGACGTTATTGAAAATAATAATGGAGACATTATTGAAGATAATAATGCAAACGAAAAAAATCATAATAATCTTGAATCTGAAGGTAAGGGTATCGTATATGATGATGTAAATAATTTACATGTTCACACAAACAGTGATAATAGTGCTCATTCGAAGAAAATAAAGGGAGCCCCCAGTAGGTTTAGTAATACAAATAATGGAAGGAAGAAACGAAGAAGGAGAAAATTCATCAATGTAGTTAATTATATAAAGAAGAAGAAAAAGAAGAAACTGATAAAAAGTATGGATAATATGGAGGTTACAGATAATTTTAAGAATGATATGAGTGATGAAAATAAACAAAGTGGTGATGAAAATAAACAAAGTGGTGATGAAAATAAACAAAGTGGTGATGAAAATAAACAAAGTGGTGATGAAAATAAACAAACTAATAATGATATTAAACAGAGTGATAATGATATTAAACAGAGTGATGATATTTACATGAATGAAGATATGAATTTGTTCAATGATTTAAATGATAACTTCGATAACAATGAATATTTCATAAACAATGGTGATAAGGATTCTCATGCTGAAGAAGAAATGGCCATAGAAAATATTCAAAGTAAAAGTATAGAAAAGGATATTTTAAATAATGAAGAGCAGGATAATAATAACATCTTTGATATTGATAATGAACTTATAGATATGAAGGATGGAAATGTAGATGAAATGGAAAGTGATGAAAAATTAAAAACTTTTGAAAAATTGGAAAGTTTGAAAAGTACAACACATTTAAACAATACCGATAATTGTGATGTAAATTTGAGTGAACAGACCAATGAAATAAATTATGATGAGGAAAAAAAAGTTAATAAAAAAACAAATCATGAAAAAATGAAGAAGAAGAAGAAGAAAAAAAAAAAAAAAAAGAAAAAGAAGAAGAAAGAAAAAAAACAAATAGATATTATGTACAAAAATTTGTCCAGACTTAATTTAAATTTGTTACTTCCAACCAAAAAAAAAGTTAAGAAATCGAAAAACTCATTTAAAAAAGAGGAAGAAAAACAAAAGAAGAAAAATAAAAAAGTTAAAAAAATCAAAGGTATTAACAAGGGGGAAAAAATAAAAAGTAATAAGAAAGAAAATAAGGACAATAATAATGATAGTAGTACAGAATGTGTTGTAGAAGGAGAAAAAGGAAAAGATTTACATGAGTTTAATAAAAATGGAAATCTTGAAGATGAACAAATGGATGTTGATATTTCTATGAATATTTCAAGTATAAATTGTGAAAGTGATAATAAAAATGTGAGTAAGGAAGGAGAGGAAGAAAAAAAAGACATAGCTGAAAACAAAGAAGAGGTGGATAAAAACAAAGAAGAGGTATATATGGACAAACATGAGATGGATTTGAACAATGAAGAGGTATATATGGACAAAAATGAGATGGATTTGAACAATGAAGAGGTATATATGGACAAACATGAGATGGATTTGAACAATGAAGAGGTATATATGGACAAACATGAAATGGATTTGAACAATGAAGAGGTATATATGGACAAACATGAAATGGATTTGAACAAAGAAGAGGTATATATGGACAAACATGAGATGGATTTGAACAATGAAGAGGTAGATAAAGAAAACGAATATGATGAAAATATACTTAGTGATAACATAATATATAATGAAAACAATTCATTTGGAAACAATAAGAACTCTTTTTTTAATAATACAAGTCCATTAAAAACAGAAATAATAAATGAAGAGGAAAATAGTTTGAACGAAATGAAAGAAGACATAAATGAATACGTTGAAATGGAAAACAAGTTGGATACGGAAAAAATAAAAGATTCAGAAAAAATAGGTGGAAAAATAGAGGTAGATAATAAAATGATTTCTCCTATTAATAGACATAATTTTTATTTAACAATTCTTGAAGGAATGAATAAGAATTTTCCTAGGCAATGGAATAAAAATAATATAACTTTATCAAAAAATCAAGGACAAATTTATAAAGGAAGGAAAGAAAAGAAAAGAAAACGTTCCTATAGAAATGATGAAAAATTACTTGATCATAGTATATTAAATGATATCAATATAAGTGACAAAATGGATGAAAGAAATGAATTATTAGAGAGTATAAAATCTAATAGTACTATAAATAATGTATTAGAAATTATAAAATATGATAATAGGAAAAAAATAAAGAAGAATGATACAAACAAGGAAATAATCAAATATGATAACTTCACATCTAAATATAATAATAAAAGTAATGATATTCAATTGAATGGTGGAATATATATAAATAAATTCAAACTTTCTTTAGATATGCCTATAAATAAATTAGCGGTATCTTCAAATCTTGGACCTCCATCATCTATAGGATCAACAGAAATACAGCCTATTCAAAAGAATTTCAACGATTTCAAAATGAATATTAACGTGTACTGTATTAGGATGGAGCCGCATGAAAAATACAGCTCATATAGCCATAAAAATAATTTAGTTGTATATATTGATAAGGGAGAAAAAATTAACATAATAATCAACATGTCAAAGACTTATGAAAAAGGTGATTTTTTTTACATACCTAGATTTTCTAACTTCCAAATAATTAATGATAGCAGATGTGATTGTGTTTTATATGTTTGTCCTTTAATTTAA(SEQ ID NO: 4) Sequence Length: 6225 bp; underlined sequencecorresponds to nucleotide sequence encoding; PfSEP-1Aantigenic fragment.

The invention is also directed in part to polynucleotides andpolypeptides shown in the Table below that are useful, for example, forantigens for vaccines against P. falciparum malaria.

Length Length of of Clone Gene Serial Plasmodb.org peptide Protein sizesize in Number Clone Name GENE ID Gene Name/Function in aa aa in bp bp 1 Clone#2 PF10_0212a PfSEP-1/Schizont 273 2074  819 6225 Version 9.2egress (2431-3249)  2 Clone#5 PF13_0197 MSP-7/Merozoite 284  351  8521056 surface protein/  (201-1052) RBC invasion  3 Clone#10 PF11_0354Schizont egress 641 2227 1923 6684 (3490-5412)  4 Clone#T108 PFB0310cMSP-4/Merozoite  79  272  238  819 surface protein/ (124-361)RBC invasion  5 Clone#T32 MAL8P1.58 Pf-PGPS/phosphatidyl 100  661  3001986 glycerophosphate (1023-1322) synthase  6 Clone#T9 PFE0040cMESA/Mature 153 1434  459 4305 Erythrocyte Surface (2080-2538) Antigen 7 Clone#TL22 PFA0620c Pf-GARP/glutamic 263  673  792 2022acid rich protein (1231-2022)  8 Clone#TL27 PFI1780w Plasmodium exported101  383  303 1152 protein (691-993)  9 Clone#TL5 PFB0100c Pf-  80  654 242 1965 KAHRP/Pathogenicity, (1309-1550) Adhesion/KnobAssociated Histidine Rich Protein 10 Clone#TL16 MAL7P1.208 RAMA/Rhoptry144  873  432 2114 Associated membrane  (953-1384) antigen/RBCinvasion/DNA mismatch repair protein 11 Clone#TL45 PF07_0033Cg4 protein/ 216  873  650 2622 parasite heat shock  (1764-2413)protein 70/ protein transport 12 PF3D7 PF13_0211 Ca⁺⁺ dep. Protein  84 568  255 1707 kinase Clone #5: MSP-7 (PF13_0197)Nucleic acid sequence of Clone #5, 852 bp (Sequence201-1,052 of gene PF13_0197) (SEQ ID NO: 5)ATTAAACAAAAAAATTGAAGAATTACAAAACAGTAAAGAAAAAAATGTACATGTATTAATTAATGGAAATTCAATTATTGATGAAATAGAAAAAAATGAAGAAAATGATGATAACGAAGAAAATAATGATGATGACAATACATATGAATTAGATATGAATGATGACACATTCTTAGGACAAAATAACGATTCACATTTTGAAAATGTTGATGATGACGCAGTAGAAAATGAACAAGAAGATGAAAACAAGGAAAAATCAGAATCATTTCCATTATTCCAAAATTTAGGATTATTCGGTAAAAACGTATTATCAAAGGTAAAGGCACAAAGTGAAACAGATACTCAATCTAAAAATGAACAAGAGATATCAACACAAGGACAAGAAGTACAAAAACCAGCACAAGGAGGAGAATCGACATTTCAAAAAGACCTAGATAAGAAATTATATAATTTAGGAGATGTTTTTAATCATGTAGTTGATATTTCAAACAAAAAGAACAAAATAAATCTCGATGAATATGGTAAAAAATATACAGATTTCAAAAAAGAATATGAAGACTTCGTTTTAAATTCTAAAGAATATGATATAATCAAAAATCTAATAATTATGTTTGGTCAAGAAGATAATAAGAGTAAAAATGGCAAAACGGATATTGTAAGTGAAGCTAAACATATGACTGATATTTTCATAAAACTATTTAAAGATAAGGAATACCATGAACAATTTAAAAATTATATTTATGGTGTTTATAGTTATGCAAAACAAAATAGTCACTTAAGTGAGAAAAAAATAAAACCAGAAGAGGAATATAAAAAATTTTTAGAATATTCATTTAATTTACTAA ACACAATSequence Length: 852 bp  Amino acid sequence of Clone #5 (SEQ ID NO: 6)LNKKIEELQNSKEKNVHVLINGNSIIDEIEKNEENDDNEENNDDDNTYELDMNDDTFLGQNNDSHFENVDDDAVENEQEDENKEKSESFPLFQNLGLFGKNVLSKVKAQSETDTQSKNEQEISTQGQEVQKPAQGGESTFQKDLDKKLYNLGDVFNHVVDISNKKNKINLDEYGKKYTDFKKEYEDFVLNSKEYDIIKNLIIMFGQEDNKSKNGKTDIVSEAKHMTDIFIKLFKDKEYHEQFKNYIYGVYSYAKQNSHLSEKKIKPEEEYKKFLEYSFNLLNTMSequence Length: 284 aa  Amino acid sequence of MSP7 gene (PF13_0197)(SEQ ID NO: 7)MKSNIIFYFSFFFVYLYYVSCNQSTHSTPVNNEEDQEELYIKNKKLEKLKNIVSGDFVGNYKNNEELLNKKIEELQNSKEKNVHVLINGNSIIDEIEKNEENDDNEENNDDDNTYELDMNDDTFLGQNNDSHFENVDDDAVENEQEDENKEKSESFPLFQNLGLFGKNVLSKVKAQSETDTQSKNEQEISTQGQEVQKPAQGGESTFQKDLDKKLYNLGDVFNHVVDISNKKNKINLDEYGKKYTDFKKEYEDFVLNSKEYDIIKNLIIMFGQEDNKSKNGKTDIVSEAKHMTEIFIKLFKDKEYHEQFKNYIYGVYSYAKQNSHLSEKKIKPEEEYKKFLEYSFNLLNTMSequence Length: 351 aa  Nucleic acid sequence of MSP7 gene (PF13_0197)(SEQ ID NO: 8)ATGAAGAGTAATATCATATTTTATTTTTCTTTTTTTTTTGTGTACTTATACTATGTTTCGTGTAATCAATCAACTCATAGTACACCAGTAAATAATGAAGAAGATCAAGAAGAATTATATATTAAAAATAAAAAATTGGAAAAACTAAAAAATATAGTATCAGGAGATTTTGTTGGAAATTATAAAAATAATGAAGAATT ATTAAACAAAAAAATTGAAGAATTACAAAACAGTAAAGAAAAAAATGTACATGTATTAATTAATGGAAATTCAATTATTGATGAAATAGAAAAAAATGAAGAAAATGATGATAACGAAGAAAATAATGATGATGACAATACATATGAATTAGATATGAATGATGACACATTCTTAGGACAAAATAACGATTCACATTTTGAAAATGTTGATGATGACGCAGTAGAAAATGAACAAGAAGATGAAAACAAGGAAAAATCAGAATCATTTCCATTATTCCAAAATTTAGGATTATTCGGTAAAAACGTATTATCAAAGGTAAAGGCACAAAGTGAAACAGATACTCAATCTAAAAATGAACAAGAGATATCAACACAAGGACAAGAAGTACAAAAACCAGCACAAGGAGGAGAATCGACATTTCAAAAAGACCTAGATAAGAAATTATATAATTTAGGAGATGTTTTTAATCATGTAGTTGATATTTCAAACAAAAAGAACAAAATAAATCTCGATGAATATGGTAAAAAATATACAGATTTCAAAAAAGAATATGAAGACTTCGTTTTAAATTCTAAAGAATATGATATAATCAAAAATCTAATAATTATGTTTGGTCAAGAAGATAATAAGAGTAAAAATGGCAAAACGGATATTGTAAGTGAAGCTAAACATATGACTGAAATTTTCATAAAACTATTTAAAGATAAGGAATACCATGAACAATTTAAAAATTATATTTATGGTGTTTATAGTTATGCAAAACAAAATAGTCACTTAAGTGAGAAAAAAATAAAACCAGAAGAGGAATATAAAAAATTCTTAGAATATTCATTTAATTTACTAAACACAA TGTAA  Sequence Length: 1056 bp  Clone#10 (PF11_0354)Nucleic acid sequence of Clone #10, 1923 bp (Sequence3490-5412 of gene PF11_0354 (SEQ ID NO: 9)GATAATGTTAATAATAATAATAATAAAGAAAGTTGTGATAATATTAAACATATGAGAACAAAAAGTTTAAATTTTGTAAGTAGAGAATCCTATGGCGAACATAAAAGTCTAGATGTTTACCAGGAATGTTATGTAAAAAATAATAAACTTATTAATAAGGTAAATGATAAAAAATATGAGGACAATAATAATTCCTATCTTAATGAAGATGATAACGCTAGTATGCAATTTTATGAAGAAACTAATAGTAATCCATATATTGTAGACCAGGAAAATAATATGAAAAATTATGTCAATAATGTTTTATATAACAACAATAGCAATTATTATGTTGATTCAAAGAATTATGATAAATCTAAAGAGAATGCAGAAAATAAATCAGATGATATATTAAATAATGAAAATATACATACCTTAAAAGATCAAAAAAAGAAAATACAAAATAATAATGAATTCATTAGTGAACAGGCTGATATAGAAAATATAAGAAATTCTCAAGAAGAAGTATATGAGAAAGAACACGAACCTTTGTGGGTAATAAATGCATCTAATGAAGAAAAGAAATCATATGAAGAATTGATATACAGCGATATGTCATCTAATCGTGTTACGAAAAATAAATATAGTGATATGAATAATGTTGAGGTATTATTAAATGAAGATAATTTATTAACTACTGAAAAATACAAGGTGCAATTAGAAAAAGAAAATAAAATGATTGATATGTATGAAACGGTAGAGGAGAATATAAATACAATTAAAACAGAAAATACGAACGACATAAATGAAGAAGTTAGAAACGAACAAAAAAGAGAAAGTATCAATCATATTAATGATACAAATATAAATCATATAATAGATGAATATCCCAATGATACATATAATTTCATAAAAGATATAGAATGTGTACATAACAATGAAAATAACATGTACAATTCTATTGAACAATATACATTTTATCATGATACACGTAATAATCATTTAGTTGATAAAAATAATCAAAATTTTATATTCGAAGAGGAAGGTTTAAATGAATTGAACTTTGAAGAAAAAAAGGTATATATAGAAAATAATACCAAGGATGATCACAAGGGAGATAGCAAAACAAGTAACTTAACATCTTTAAGGAATACCATATGTAAAAGTGAAAACGATCATAATGAAAAAAATGAAAACACATATGTGGTTAGAAAAGGCGAAAAAGGAATTAAACGTAAGGTTTCCATGAAGAAAAGAAATGAAAAGCTAAATGAAGAAAATTATATTAATAATATATACGATAAAATGGATAACCATAGACAAAATGATATTACAAAAAAAGAAAATGACGAAGAAAATTATATTTTGTACAACAACGTAAAGGTTAATTATGATGAATATATAGAAAATGGAAATAAAATAAAAATAACGGAAGAATCATTAAATGTCTTTTATAAAGAAAATCAAAATGAGGAAGATTCTTCTACAAAAAAGTTGAATAGTACAAGTAAAATAAAACGTGCAAACAAAGGGAAAACAAAAAAAAAGAATGTTATCACAAGGGTACATAAAACAAAACAAAAAATTGAATATGTTACAAATAGTTTTAATAAATCTTCCAAAGGTGAAAATTCAGAAATAGGAAAAATTGGAGGTAGGAGTAAATCATTATTAACACACAGCAAGAAAGTTAGTGAACGAAATAAAAATAAAATAGAAAAAATTAATGATACAAATTCAAAGATAATAAAAGGAAAAAAGAGTAATAGCCAAAGCAAACTTGGGAAGGATACAAAAATTAGAGGGAAATCAAAAACTGGGGAATATATAAAAAATAAAGATTTAAGAAAAAAATCTAACGAAAAAAACAAAACAGTGATGGATAATATAAATACTATAAATAATTCTTCAGTATCTAACCTAAAAAGCAAAAAACATAAATTG  Sequence Length: 1923 Amino acid sequence of Clone # 10, (PF11_0354) (SEQ ID NO: 10)DNVNNNNNKESCDNIKHMRTKSLNFVSRESYGEHKSLDVYQECYVKNNKLINKVNDKKYEDNNNSYLNEDDNASMQFYEETNSNPYIVDQENNMKNYVNNVLYNNNSNYYVDSKNYDKSKENAENKSDDILNNENIHTLKDQKKKIQNNNEFISEQADIENIRNSQEEVYEKEHEPLWVINASNEEKKSYEELIYSDMSSNRVTKNKYSDMNNVEVLLNEDNLLTTEKYKVQLEKENKMIDMYETVEENINTIKTENTNDINEEVRNEQKRESINHINDTNINHIIDEYPNDTYNFIKDIECVHNNENNMYNSIEQYTFYHDTRNNHLVDKNNQNFIFEEEGLNELNFEEKKVYIENNTKDDHKGDSKTSNLTSLRNTICKSENDHNEKNENTYVVRKGEKGIKRKVSMKKRNEKLNEENYINNIYDKMDNHRQNDITKKENDEENYILYNNVKVNYDEYIENGNKIKITEESLNVFYKENQNEEDSSTKKLNSTSKIKRANKGKTKKKNVITRVHKTKQKIEYVTNSFNKSSKGENSEIGKIGGRSKSLLTHSKKVSERNKNKIEKINDTNSKIIKGKKSNSQSKLGKDTKIRGKSKTGEYIKNKDLRKKSNEKNKTVMDNINTINNSSVSNLKSKKHKL,Sequence Length: 641  Amino acid sequence of PF11_0354 (SEQ ID NO: 11)MRSKSISYFLFFKKNKKKNDSCDSVIISSNKNLSIQLSKGEDDEKNEINEEKSYIKNEDVYKKEKLKKKKENKENNKKKDKNEVVYDYHDISNDATSDYVNNYKVYEMNTCNIKKKRESFFKKINILQKYKNYKIRKAASTFHTIGHKTSFSGTDDEIENNQKKQKKYKIKISEWKDDKSHTFHKKNDILVFDKMDKNKKFKIDNNKNNQINIDNEERVNKNYPMATNVQNFNIKYTSIDVTNDEYIIDSNKPEGSIMSTDKKNNKLNYNNDTYDVDKSSDINKLGNIKKNKFDIITKTTHNINNNVNNIHNYMMYTNKENIKININHGNLNGREQNNYDEERKANVYEIFENAKKLEPNNININTEEHIHISEPSIPFDMKDHKNDINEKDIILKLMYNNNGIYFDDDDENHKNLLYKNKDTHVKHLNNKFNHNFIIYNDREEGVNQKHAQKKLKKKNTILNKNENEDINHNSFKRPLSNTNICYKDKDDKIKNGSNKYDILNNDYSNEHEKNKYNDHITKNKRNQSANEVKSNNNDNHNNKKNNNFNININDSYSTNINRNQNVMINDVNDVIKDPNMQENTQGDDEGGIINKYLINPIYNLFLRANEEIQNSNSTNNKLKMNNITKSYTNELQKTYKSMYDINDISNKRKINNKDIRGTNLYNTKLCNNKLYNSNPYNMIPYNINTYNNNNNNKETCTSINIKHSENKYPFNKSHVNSYMKNTNHLPHRNAITSNNRNNEEYEKEKEKDRNITNGNNNYLVEYNNSCIPPPLKKMIPIDGVRNKSINKLNNVTNTQRTSSVSYTNKNIDENSFDMPIINGIRESKYISNNNNINGNSIGFNSSKLDNYHHQSMNVNESYPLKNMMKNNYIEHNYDDKNNIFLVKNYEDTYSNIHNGIHENSMLKNYNLKKACTFHGYSRNHQKNMYTEENLNINQKKNYSHYHNNGTVLKPLVNTNNVAVNEFADINLSAQKRLHSLKSMGYEDKSMENYRNKIYNNINNNNNNNNDNNIYNDNEYCQYNNSYCFDHSDLKNMFPLNHQNSKLLTHSNNKNSFFNGINVESKHHLANPEIKTFAHNSYPILNQGLINCNPLQCLGYDSNQRNKHNVVYIKKNEYLNKNIGSIINVLKREGLRKISTHNGKFESFSNMDNKNVYMEGLNIQ DNVNNNNNKESCDNIKHMRTKSLNFVSRESYGEHKSLDVYQECYVKNNKLINKVNDKKYEDNNNSYLNEDDNASMQFYEETNSNPYIVDQENNMKNYVNNVLYNNNSNYYVDSKNYDKSKENAENKSDDILNNENIHTLKDQKKKIQNNNEFISEQADIENIRNSQEEVYEKEHEPLWVINASNEEKKSYEELIYSDMSSNRVTKNKYSDMNNVEVLLNEDNLLTTEKYKVQLEKENKMIDMYETVEENINTIKTENTNDINEEVRNEQKRESINHINDTNINHIIDEYPNDTYNFIKDIECVHNNENNMYNSIEQYTFYHDTRNNHLVDKNNQNFIFEEEGLNELNFEEKKVYIENNTKDDHKGDSKTSNLTSLRNTICKSENDHNEKNENTYVVRKGEKGIKRKVSMKKRNEKLNEENYINNIYDKMDNHRQNDITKKENDEENYILYNNVKVNYDEYIENGNKIKITEESLNVFYKENQNEEDSSTKKLNSTSKIKRANKGKTKKKNVITRVHKTKQKIEYVTNSFNKSSKGENSEIGKIGGRSKSLLTHSKKVSERNKNKIEKINDTNSKIIKGKKSNSQSKLGKDTKIRGKSKTGEYIKNKDLRKKSNEKNKTVMDNINTINNSSVSNLKSKKHKL KKKKKKNISMENINKNITNEFCSMERKGTVLLSNMSIKKIDNANSCTLNEPLEENTLNYESNNNCSNSNLSKDKEKDRNILCNKYYSDEETNSLNKMYTSNIPEISNYYKEIQAINYILSNINNPNFLNSLELNDLINIEKKFINENIYINKQIIACNVKNEKSNDEMVEKNERKVDEEKGEDEQEIKAKENNNKEENQDNENNNKEENHDNENNNKEENQDNENNNKEENQDNENNNKEENQDNENNNKEENQKNENGIIYDSRFSIIYLEHDLIYLKKNNLKVILNVLLSNVYCFFEIKLTIILLNFFISNNCQWSFSLFPLSLINKLIHKFSLKINKKVPKYKLENMNINSPNIPYTYLFICDGSNYLCINDNSLNNEVYENKMKLNNIIGYYHYINLNRLTYYLEKVNANFVYNHHIYE,Sequence Length: 2227  Coding Nucleic acid sequence gene PF11_0354(SEQ ID NO: 12)ATGAGATCGAAATCCATTTCGTATTTCTTATTTTTTAAAAAAAACAAAAAGAAAAATGATTCTTGTGATAGTGTCATAATATCTAGCAATAAGAATTTATCCATTCAATTATCGAAAGGTGAGGATGATGAAAAAAATGAAATAAATGAGGAAAAGAGTTATATAAAAAATGAAGATGTATATAAAAAGGAAAAATTAAAAAAGAAGAAAGAAAACAAGGAAAATAATAAAAAGAAAGATAAAAATGAAGTAGTATATGATTATCATGACATTTCAAATGATGCTACTAGTGATTATGTTAATAATTATAAAGTATATGAAATGAATACTTGTAATATAAAAAAGAAGAGAGAAAGTTTTTTTAAAAAAATTAATATTTTACAAAAATATAAAAATTACAAAATTAGAAAGGCAGCTAGTACCTTTCATACCATAGGACATAAAACATCTTTTTCTGGTACAGATGATGAAATAGAAAATAATCAAAAGAAACAAAAAAAATATAAAATAAAAATTTCTGAATGGAAGGATGATAAATCACATACTTTTCATAAAAAAAATGACATATTGGTATTTGATAAGATGGATAAAAATAAAAAATTTAAAATTGATAACAACAAAAACAATCAAATTAATATAGATAATGAAGAAAGAGTTAATAAAAATTATCCTATGGCTACTAATGTACAAAATTTTAATATAAAATATACATCAATAGATGTAACAAATGACGAATATATTATAGATTCTAATAAACCTGAAGGTTCTATTATGTCTACAGATAAAAAGAATAATAAACTTAATTATAATAATGATACATATGATGTAGACAAAAGCTCTGATATAAATAAGTTAGGTAATATAAAAAAGAATAAATTTGATATTATTACTAAAACAACACATAATATTAATAATAATGTAAATAATATACATAATTATATGATGTATACAAATAAAGAAAATATAAAAATAAATATAAATCATGGAAATCTAAATGGAAGAGAACAAAACAATTATGATGAAGAAAGGAAAGCAAATGTTTATGAAATATTTGAAAATGCAAAAAAATTAGAACCTAATAATATTAATATCAACACAGAAGAACATATTCATATTAGTGAACCCAGCATACCATTTGATATGAAGGATCATAAAAATGATATAAATGAAAAAGATATAATATTAAAATTGATGTATAACAATAACGGTATTTATTTTGATGATGATGATGAAAATCACAAGAATTTATTATACAAAAATAAAGATACACATGTAAAACATTTAAATAATAAATTTAACCATAATTTTATTATATATAATGATCGCGAAGAAGGGGTAAATCAGAAACACGCACAAAAAAAATTAAAAAAAAAAAATACTATTCTTAACAAAAACGAAAATGAAGATATTAATCATAATAGTTTCAAAAGACCTTTATCTAATACGAATATATGTTATAAGGACAAAGATGATAAAATTAAAAATGGTTCTAATAAGTATGATATATTAAATAATGACTATTCTAATGAACACGAAAAAAATAAATATAATGATCATATAACAAAAAATAAAAGAAATCAATCAGCAAATGAAGTAAAATCTAATAATAATGATAACCACAATAATAAAAAAAATAATAATTTTAATATTAATATTAATGATTCATATTCTACAAATATAAATAGAAACCAAAATGTGATGATAAATGATGTAAACGATGTTATTAAGGATCCAAATATGCAGGAAAATACACAAGGTGATGACGAAGGTGGTATTATAAACAAATATTTAATTAACCCTATTTACAATTTATTTCTACGTGCTAATGAAGAAATACAAAATTCAAATAGTACAAACAATAAATTAAAAATGAATAATATAACAAAAAGTTATACAAACGAACTACAAAAGACATATAAAAGTATGTACGATATAAATGATATATCAAATAAGAGAAAAATTAATAATAAAGATATACGTGGAACTAATTTGTATAACACCAAATTATGTAATAATAAATTATATAATTCGAATCCATATAATATGATTCCATATAATATAAACACATATAATAATAATAATAATAATAAGGAAACTTGTACCAGCATAAATATCAAACATTCCGAAAATAAATATCCCTTCAATAAATCTCATGTAAACTCATATATGAAAAATACAAATCATCTTCCTCATAGAAATGCGATTACATCAAATAATAGAAACAATGAAGAATATGAGAAAGAAAAAGAAAAAGATCGTAACATTACTAATGGGAACAATAATTATTTGGTTGAATATAATAATTCTTGTATACCTCCACCACTCAAAAAAATGATACCAATAGATGGTGTGAGAAATAAAAGTATAAATAAATTAAATAATGTAACTAATACGCAACGTACATCAAGTGTTTCATATACGAATAAGAATATTGATGAGAATTCGTTTGATATGCCTATAATAAATGGAATAAGAGAATCTAAATATATAAGTAATAATAATAATATTAATGGTAATTCCATTGGTTTTAATTCATCTAAGTTAGATAATTATCATCACCAATCTATGAATGTGAATGAATCTTATCCTCTAAAAAATATGATGAAAAATAATTATATTGAACATAATTATGATGATAAAAATAATATTTTCCTTGTTAAAAATTATGAAGATACATATTCAAATATTCATAATGGCATACATGAAAATAGCATGCTAAAAAATTATAATTTAAAAAAAGCGTGCACTTTTCATGGGTACTCTAGAAATCACCAAAAAAATATGTATACGGAAGAAAATTTAAATATTAATCAAAAAAAGAATTATAGTCATTATCATAATAATGGAACGGTATTAAAACCTTTGGTAAATACTAATAATGTTGCAGTGAACGAATTTGCAGATATTAATTTATCGGCTCAAAAAAGATTACATAGTTTAAAAAGTATGGGGTACGAGGATAAGAGTATGGAAAATTACAGAAACAAAATATACAACAACATCAATAATAATAATAATAATAATAATGATAATAATATATATAATGATAATGAATATTGTCAGTATAATAATAGTTATTGTTTCGATCATAGTGATTTAAAAAATATGTTTCCATTAAATCATCAGAATAGCAAGTTATTAACACATAGTAATAATAAAAATTCATTTTTTAACGGAATAAATGTAGAATCGAAACATCATTTAGCAAATCCTGAAATAAAAACATTTGCACACAATAGTTATCCTATATTAAATCAAGGTTTAATAAATTGTAACCCCTTACAATGCTTGGGTTATGATTCAAATCAAAGGAATAAGCATAATGTAGTATACATAAAAAAAAATGAATACCTTAATAAAAACATTGGCTCTATTATAAATGTTCTTAAAAGAGAAGGACTAAGAAAAATTTCTACACATAATGGAAAATTCGAATCATTTAGTAATATGGATAATAAAAATGTATATATGGAAGGACTAAACATACAA GATAATGTTAATAATAATAATAATAAAGAAAGTTGTGATAATATTAAACATATGAGAACAAAAAGTTTAAATTTTGTAAGTAGAGAATCCTATGGCGAACATAAAAGTCTAGATGTTTACCAGGAATGTTATGTAAAAAATAATAAACTTATTAATAAGGTAAATGATAAAAAATATGAGGACAATAATAATTCCTATCTTAATGAAGATGATAACGCTAGTATGCAATTTTATGAAGAAACTAATAGTAATCCATATATTGTAGACCAGGAAAATAATATGAAAAATTATGTCAATAATGTTTTATATAACAACAATAGCAATTATTATGTTGATTCAAAGAATTATGATAAATCTAAAGAGAATGCAGAAAATAAATCAGATGATATATTAAATAATGAAAATATACATACCTTAAAAGATCAAAAAAAGAAAATACAAAATAATAATGAATTCATTAGTGAACAGGCTGATATAGAAAATATAAGAAATTCTCAAGAAGAAGTATATGAGAAAGAACACGAACCTTTGTGGGTAATAAATGCATCTAATGAAGAAAAGAAATCATATGAAGAATTGATATACAGCGATATGTCATCTAATCGTGTTACGAAAAATAAATATAGTGATATGAATAATGTTGAGGTATTATTAAATGAAGATAATTTATTAACTACTGAAAAATACAAGGTGCAATTAGAAAAAGAAAATAAAATGATTGATATGTATGAAACGGTAGAGGAGAATATAAATACAA TTAAAACAGAAAATACGAACGACATAAATGAAGAAGTTAGAAACGAACAAAAAAGAGAAAGTATCAATCATATTAATGATACAAATATAAATCATATAATAGATGAATATCCCAATGATACATATAATTTCATAAAAGATATAGAATGTGTACATAACAATGAAAATAACATGTACAATTCTATTGAACAATATACATTTTATCATGATACACGTAATAATCATTTAGTTGATAAAAATAATCAAAATTTTATATTCGAAGAGGAAGGTTTAAATGAATTGAACTTTGAAGAAAAAAAGGTATATATAGAAAATAATACCAAGGATGATCACAAGGGAGATAGCAAAACAAGTAACTTAACATCTTTAAGGAATACCATATGTAAAAGTGAAAACGATCATAATGAAAAAAATGAAAACACATATGTGGTTAGAAAAGGCGAAAAAGGAATTAAACGTAAGGTTTCCATGAAGAAAAGAAATGAAAAGCTAAATGAAGAAAATTATATTAATAATATATACGATAAAATGGATAACCATAGACAAAATGATATTACAAAAAAAGAAAATGACGAAGAAAATTATATTTTGTACAACAACGTAAAGGTTAATTATGATGAATATATAGAAAATGGAAATAAAATAAAAATAACGGAAGAATCATTAAATGTCTTTTATAAAGAAAATCAAAATGAGGAAGATTCTTCTACAAAAAAGTTGAATAGTACAAGTAAAATAAAACGTGCAAACAAAGGGAAAACAAAAAAAAAGAATGTTATCACAAGGGTACATAAAACAAAACAAAAAATTGAATATGTTACAAATAGTTTTAATAAATCTTCCAAAGGTGAAAATTCAGAAATAGGAAAAATTGGAGGTAGGAGTAAATCATTATTAACACACAGCAAGAAAGTTAGTGAACGAAATAAAAATAAAATAGAAAAAATTAATGATACAAATTCAAAGATAATAAAAGGAAAAAAGAGTAATAGCCAAAGCAAACTTGGGAAGGATACAAAAATTAGAGGGAAATCAAAAACTGGGGAATATATAAAAAATAAAGATTTAAGAAAAAAATCTAACGAAAAAAACAAAACAGTGATGGATAATATAAATACTATAAATAATTCTTCAGTATCTAACCTAAAAAGCAAAAAACATAAATTG AAAAAAAAAAAAAAAAAAAATATATCTATGGAAAATATAAATAAAAATATAACAAATGAATTTTGTTCTATGGAAAGAAAAGGAACCGTTCTATTATCTAATATGAGTATTAAGAAGATTGATAATGCAAATAGTTGTACATTAAATGAACCATTAGAGGAAAATACCTTAAATTATGAAAGTAATAATAACTGTAGTAATAGTAATTTATCTAAGGATAAAGAAAAAGATAGAAATATATTGTGTAATAAATATTATAGTGATGAGGAAACAAACTCTTTAAACAAAATGTATACATCGAATATACCAGAAATAAGTAATTATTATAAGGAAATTCAAGCAATTAATTACATATTAAGTAATATTAATAATCCAAATTTTTTAAATTCCCTCGAACTGAATGATTTAATAAATATTGAAAAAAAATTTATTAACGAAAATATATATATTAATAAGCAGATAATAGCCTGTAATGTAAAAAATGAAAAATCAAATGATGAGATGGTCGAGAAAAATGAACGCAAAGTGGATGAAGAAAAAGGAGAAGACGAACAAGAAATAAAAGCAAAGGAAAATAATAATAAAGAAGAAAACCAAGATAATGAAAATAATAATAAAGAAGAAAACCATGATAATGAAAATAATAATAAAGAAGAAAATCAAGATAATGAAAATAATAATAAAGAAGAAAACCAAGATAATGAAAATAATAATAAAGAAGAAAATCAAGATAATGAAAATAATAATAAAGAAGAAAACCAAAAAAATGAAAATGGTATTATTTATGATAGCAGGTTTAGTATTATCTATTTAGAACACGATTTAATATATTTAAAAAAAAATAATTTAAAAGTGATACTTAATGTTTTGCTGTCAAATGTGTATTGCTTTTTTGAAATTAAATTAACCATAATATTGTTAAATTTCTTTATATCTAATAATTGTCAATGGAGTTTCAGTTTATTTCCCCTTTCATTAATTAATAAATTAATACATAAATTCAGTTTAAAGATAAATAAGAAAGTTCCTAAATATAAATTGGAAAATATGAATATTAACTCACCAAATATTCCATATACATATCTTTTTATATGTGATGGAAGTAACTATTTATGTATTAATGACAATTCATTAAATAACGAGGTATATGAAAACAAGATGAAATTGAACAATATCATTGGATATTACCATTATATTAATTTGAATAGATTAACATATTATTTAGAAAAGGTAAATGCTAATTTTGTTTATAACCATCATATATATGAATAA, Sequence Length: 6684 bp Clone # T108: MSP-4 (PFB0310c)Nucleic acid sequence of Clone# T108, 238 bp (Sequence124-361 of gene PFB0310c 1-819) (SEQ ID NO: 13)AGAATTCTAGGGGAAGAAAAACCAAATGTGGACGGAGTAAGTACTAGTAATACTCCTGGAGGAAATGAATCTTCAAGTGCTTCCCCCAATTTATCTGACGCAGCAGAAAAAAAGGATGAAAAAGAAGCTTCTGAACAAGGAGAAGAAAGTCATAAAAAAGAAAATTCCCAAGAAAGCGCGAATGGTAAGGATGATGTTAAAGAAGAAAAAAAAACTAATGAAAAAAAAGATGATGGAA  Sequence Length: 238 bp Amino acid sequence of Clone# T108 (SEQ ID NO: 14)RILGEEKPNVDGVSTSNTPGGNESSSASPNLSDAAEKKDEKEASEQGEESHKKENSQESANGKDDVKEEKKTNEKKDDG Sequence Length: 79 aa Amino acid sequence of PFB0310c (MSP-4) (SEQ ID NO: 15)MWIVKFLIVVHFFIICTINFDKLYISYSYNIVPENGRMLNM R ILGEEKPNVDGVSTSNTPGGNESSSASPNLSDAAEKKDEKEASEQGEESHKKENSQESANGKDDVKEEKKTNEKKDDG KTDKVQEKVLEKSPKESQMVDDKKKTEAIPKKVVQPSSSNSGGHVGEEEDHNEGEGEHEEEEEHEEDDDDEDDDTYNKDDLEDEDLCKHNNGGCGDDKLCEYVGNRRVKCKCKEGYKLEGIECVELLSLASSSLNLIFNSFITIFVVILLIN,  Sequence Length: 272 aaCoding Nucleotide Sequence of PFB0310c (MSP-4) (SEQ ID NO: 16)ATGTGGATAGTTAAATTTTTAATAGTAGTTCATTTTTTTATAATTTGTACCATAAACTTTGATAAATTGTATATCAGTTATTCTTATAATATAGTACCAGAAAATGGAAGAATGT TAAATATGAGAATTCTAGGGGAAGAAAAACCAAATGTGGACGGAGTAAGTACTAGTAATACTCCTGGAGGAAATGAATCTTCAAGTGCTTCCCCCAATTTATCTGACGCAGCAGAAAAAAAGGATGAAAAAGAAGCTTCTGAACAAGGAGAAGAAAGTCATAAAAAAGAAAATTCCCAAGAAAGCGCGAATGGTAAGGATGATGTTAAAGAAGAAAAAAAAACTAATGAAAAAAAAGATGATG GAAAAACAGACAAGGTTCAAGAAAAGGTTCTAGAAAAGTCTCCAAAAGAATCCCAAATGGTTGATGATAAAAAAAAAACTGAAGCTATCCCTAAAAAGGTAGTTCAACCAAGTTCATCAAATTCAGGTGGCCATGTTGGAGAGGAGGAAGACCACAACGAAGGAGAAGGAGAACATGAAGAGGAGGAAGAACATGAAGAAGATGACGATGACGAAGATGATGATACTTATAATAAGGACGATTTGGAAGATGAAGATTTATGTAAACATAATAATGGGGGTTGTGGAGATGATAAATTATGTGAATATGTTGGGAATAGAAGAGTAAAATGTAAATGTAAAGAAGGATATAAATTAGAAGGTATTGAATGTGTTGAATTATTATCCTTAGCATCTTCTTCTTTAAATTTAATTTTTAATTCATTTATAACAATATTTGTTGTTATATTGTTAATAAATTAA,  Sequence Length: 819 bp  Clone # T32: Pf-PGPS(MAL8P1.58)Nucleic acid sequence of Clone#T32, 300 bp (Sequence 1,023-1,3,22 of gene MAL8P1.58 (Pf-PGPS) 1-1986 (SEQ ID NO: 17)TTCTTTTATCCTTTATTTGAAAAAAATAAAAGCATTTTAGTACTTGAACTTTCCTTGCAGTGTGGATTTTCCATACCTCCAATATATGATGAAACAGATATGTTAGAAAACTTATTAAAAAATATCGAAAAATATGATCAAAGCTTAGTTATTTCTTCGGGATATTTAAACTTCCCAATGAATTTTCTTAAATTAATTAGAAATATATATATCAACGTTATGCAAAAAAAAAATGGTATTTTACAATTAATCACAGCGTCCCCATGCGCTAATATTTTTTATAAATC TAAAGGGATATCTSequence Length: 300 bp Amino acid sequence of Clone#T32 (SEQ ID NO: 18)FFYPLFEKNKSILVLELSLQCGFSIPPIYDETDMLENLLKNIEKYDQSLVISSGYLNFPMNFLKLIRNIYINVMQKKNGILQLITASPCANSFYKSKGIS,  Sequence Length: 100 Amino acid sequence of MAL8P1.58 (PfPGPS) (SEQ ID NO: 19)MALKFVIHEPKAKLLFTPKEFFNTLNDIFKNSQNRIVISCLYMGIGELEKELIDSIKKNVNIKDLKVDILLDRQRGTRLEGKFNESSVSILSELFKCSDNINISLFHNPLLGPILYNILPPRANEAIGVMHMKIYIGDNILMLSGANLSDSYLRNRQDRYFVIENKFLADSIHNIINTIQGMSFTLNRDLTIKWENDLMNPLIDAYVFREQYYRRIRFMLQGIQKHISQYNKNYSYNNYYKNIKNDPINDKTYIYNNQNNNKYSYTSNEFRMLNSFSTDIFDKDTYNNKNQKNNHKKENMETHTLLDTNHGTCDSTINLLNNNQNENHTNNLFTYLNEKDE FFYPLFEKNKSILVLELSLQCGFSIPPIYDETDMLENLLKNIEKYDQSLVISSGYLNFPMNFLKLIRNIYINVMQKKNGILQLITASPCANSFYKSKGIS YYIPSSYSAMANVCIEYITKNLTNFLKKVNGQNVSEQNDISNQKIYIEYYKPSWTFHSKGIWIMDNMKSMKNVSNDNDNDNDNNNNDNNNNNNINNNEFHSAKKYEQNVNNSPNVKNNLNKSEYFNNENFDKNIDEENDYYDNLPWCTVIGSSNYGYRAKYRDLEMSFIIKTNDYNLRCQLKKELNIIYESSHFVQVDELKLRYAFWLKFLVKYIFKWLL,Sequence Length: 661 Coding Nucleic acid sequence of gene MAL8P1.58 (PfPGPS) 1-1986(SEQ ID NO: 20)ATGGCTCTGAAGTTTGTCATTCATGAACCTAAAGCAAAATTATTATTTACTCCTAAAGAATTTTTTAATACCTTAAATGACATTTTTAAGAACTCACAAAATCGTATTGTGATTAGCTGTTTATATATGGGAATAGGAGAATTAGAAAAAGAATTAATAGATAGTATAAAAAAGAATGTGAATATAAAAGATTTAAAAGTTGATATATTATTAGATAGACAAAGAGGTACAAGACTAGAAGGGAAATTTAATGAAAGTTCAGTTAGTATTTTATCAGAACTTTTTAAATGTTCAGATAATATTAATATAAGCTTATTTCATAATCCTTTATTAGGTCCTATACTTTATAATATCTTACCTCCTAGAGCAAATGAAGCTATAGGTGTAATGCATATGAAAATTTATATTGGGGATAATATTCTAATGTTATCAGGAGCCAATTTAAGTGATAGCTATTTACGAAATAGACAAGATAGATATTTTGTTATTGAAAATAAATTCTTAGCTGATTCTATTCATAATATTATTAATACCATACAAGGTATGTCATTTACTCTAAATCGAGATTTAACCATAAAGTGGGAAAATGATTTAATGAACCCACTTATAGATGCTTACGTATTTCGTGAACAATATTATAGAAGAATACGTTTTATGTTACAAGGAATTCAAAAACATATTTCACAATATAATAAAAATTATTCATATAATAATTATTATAAAAATATAAAAAATGATCCAATAAATGATAAGACATATATTTATAATAATCAAAATAACAATAAATATAGTTATACATCAAACGAATTTCGCATGTTAAATTCTTTCAGTACAGATATATTCGATAAAGATACTTATAATAATAAAAACCAAAAAAATAATCATAAAAAAGAAAATATGGAAACACATACTTTATTAGATACTAATCATGGAACATGTGATTCAACAATTAATCTTCTAAATAATAATCAAAATGAAAACCATACAAATAATTTATTTACATATCTAAATGAAAAAGATGAATTCTTTTATCCATTATTTGAAAAAAATAAAAGCATTTTAGTACTTGAACTTTCCTTGCAGTGTGGATTTTCCATACCTCCAATATATGATGAAACAGATATGTTAGAAAACTTATTTAAAAAATATCGAAAAATATGATCAAAGCTTAGTTATTTCTTCGGGATATTTAAACTTCCCAATGAATTTTCTTAAATTAATTAGAAATATATATATCAACGTTATGCAAAAAAAAAATGGTATTTTACAATTAATCACAGCGTCACCATGCGCTAATAGTTTTTATAAATCTAAAGGGATATCT TATTATATACCAAGTTCATATTCAGCTATGGCTAATGTGTGTATTGAATATATTACCAAAAATTTAACCAATTTTCTAAAAAAAGTAAATGGACAAAATGTTTCTGAACAAAATGATATTTCAAATCAAAAAATATATATTGAATATTACAAACCTTCATGGACATTTCATTCGAAAGGTATATGGATAATGGACAATATGAAAAGTATGAAAAATGTGAGTAATGATAATGATAATGATAATGATAATAATAATAATGATAATAATAATAATAATAATATTAATAATAATGAATTTCATTCAGCTAAAAAATATGAACAAAATGTTAATAACTCACCAAATGTAAAAAATAACCTGAACAAGTCAGAATATTTTAACAACGAAAATTTTGATAAGAATATTGATGAAGAGAATGATTATTATGATAATTTACCCTGGTGTACAGTGATTGGAAGTTCTAATTATGGGTATAGAGCAAAATATAGAGATTTGGAGATGAGTTTTATAATAAAAACAAATGATTATAATTTGAGGTGTCAGTTAAAGAAAGAATTAAATATAATATATGAGTCATCTCATTTTGTACAAGTGGATGAATTGAAATTACGATATGCTTTTTGGTTAAAATTTTTAGTGAAATATATATTCAAATGGCTTTTATAA  Sequence Length: 1986 bpClone #T9: Mature parasite-infected erythrocyte surface antigen,erythrocyte membrane protein 2 (MESA)Nucleic acid sequence of Clone# T9, 459 bp (Sequence2,080-2,538 of PFE0040c (MESA) (SEQ ID NO: 21)GTAAAAGAAGGAATTAAAGAAAATGATACTGAAAATAAAGATAAAGTGATAGGACAAGAAATAATAACTGAAGAAGTAAAAGAAGGAATTAAAGAAAATGATACTGAAAATAAAGATAAAGTGATAGGACAAGAAATAATAACTGAAGAAGTAAAAAAAGAAATTGAAAAACAAGAAGAAAAAGGAAATAAAGAAAATATTCTTGAAATTAAAGATATAGTAATTGGACAAGAAGTAATAATAGAAGAAGTAAAAAAAGTAATTAAAAAAAAAGTAGAAAAAGGAATTAAAGAAAATCATACTGAAAGTAAAGATAAAGTGATAGGACAAGAAATAATAGTTGAAGAAGTAAAAGAAGAAATTGAAAAACAAGTAGAAGAAGGAATTAAAGAAAATGATACTGAAAGTAAAGATAAAGTGATAGGACAAGAAGTGATAAAAGGAGATGTTAATGAAGAA  Sequence Length: 459 bp Amino acid sequence of Clone# T9 (SEQ ID NO: 22)VKEGIKENDTENKDKVIGQEIITEEVKEGIKENDTENKDKVIGQEIITEEVKKEIEKQEEKGNKENILEIKDIVIGQEVIIEEVKKVIKKKVEKGIKENHTESKDKVIGQEIIVEEVKEEIEKQVEEGIKENDTESKDKVIGQEVIKGDVNEE  Sequence Length 153 aa Amino acid sequence of PFE0040c (MESA) (SEQ ID NO: 23)MEVICRNLCYDKKNNMMENEGNKVKKVYNNSSLKKYMKFCLCTIICVFLLDIYTNCESPTYSYSSIKNNNDRYVRILSETEPPMSLEEIMRTFDEDHLYSIRNYIECLRNAPYIDDPLWGSVVTDKRNNCLQHIKLLEMQESERRKQQEEENAKDIEEIRKKEKEYLMKELEEMDESDVEKAFRELQFIKLRDRTRPRKHVNVMGESKETDESKETDESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETGESKETRIYEETKYNKITSEFRETENVKITEESKDREGNKVSGPYENSENSNVTSESEETKKLAEKEENEGEKLGENVNDGASENSEDPKKLTEQEENGTKESSEETKDDKPEENEKKADNKKKSKKKKKSFFQMLGCNFLCNKNIETDDEEETLVVKDDAKKKHKFLREANTEKNDNEKKDKLLGEGDKEDVKEKNDEQKDKVLGEGDKEDVKEKNDEQKDKVLGEGDKEDVKEKNDGKKDKVIGSEKTQKEIKEKVEKRVKKKCKKKVKKGIKENDTEGNDKVKGPEIIIEEVKEEIKKQVEDGIKENDTEGNDKVKGPEIITEEVKEEIKKQVEEGIKENDTEGNDKVKGPEIITEEVKEEIKKQVEEGIKENDTESKDKLIGQEIITEEVKEGIKENDTENKDKVIGQEIITEEVKEGIKENDTENKDKVIGQEIITEEVKKEIEKQEEKGNKENILEIKDIVIGQEVIIEEVKKVIKKKVEKGIKENHTESKDKVIGQEIIVEEVKEEIEKQVEEGIKENDTESKDKVIGQEVIKGDVNEEGPENKDKVTKQEKVKEVKKEVKKKVKKRVKKRNNKNERKDNVIGKEIMKEDVNEKDTANKDKEIEQEKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKDTESKDKEIEQEKEKEEVKEVKEKDTENKDKVIGQEIIIEEIKKEVKKRVKKRNNKNENKDNVIVQEIMNEDVNEKDTANKDKVIEQEKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKEEVKEKDTESKDNVIVQEIMNEDVNEKDTESKDKMIGKEVIIEEVKEEVKKRVNKEVNKRVNRRNRKNERKDVIEQEIVSEEVNEKDTKNNDKKIGKRVKKPIDDCKKEREVQEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESEEESDEEKNTSGLVHRRNCKKEKKYNNGELEEYYKEKQNEEYFDEEYIIQSKEHNTLNTFPNMALNEDFRREFHNILSIHEDTDLMELKRILYNLFLEYNPHMNNKQKAELDKKFSEMNVVHQILNYEERIRMYEENAARGRLNTVILDPIITFNVIFGDDT MFKFIDE Sequence Length: 1434 aa  Coding Nucleotide sequence of PFE0040c (MESA)(SEQ ID NO: 24) TGGAGGTAATTTGTAGAAATTTATGCTACGATAAGAAAAATAATATGATGGAAAATGAAGGGAACAAAGTGAAAAAAGTGTATAATAATTCTTCTTTAAAGAAATATATGAAGTTTTGTTTATGCACTATAATATGTGTTTTTTTATTAGATATCTATACGAATTGTGAATCACCCACCTATTCATACAGTTCAATAAAGAATAATAATGACAGATATGTAAGAATTTTAAGTGAAACTGAACCACCGATGAGTTTAGAGGAAATAATGAGAACATTTGATGAAGATCATCTATATTCTATAAGAAACTATATTGAATGTTTAAGAAACGCTCCATATATCGATGATCCTTTGTGGGGTTCGGTTGTTACAGATAAACGTAATAATTGTCTTCAGCATATTAAATTATTGGAAATGCAAGAATCCGAAAGAAGAAAACAACAAGAAGAGGAGAATGCTAAGGATATTGAAGAAATAAGAAAGAAAGAAAAAGAATACCTTATGAAAGAATTAGAAGAAATGGATGAATCCGATGTAGAAAAGGCATTTAGAGAATTACAATTTATTAAGTTAAGAGATAGAACTAGACCTAGAAAACATGTGAATGTAATGGGAGAATCTAAGGAAACAGATGAATCTAAGGAAACAGATGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACTGGTGAATCTAAGGAAACAAGAATATATGAGGAAACAAAATATAACAAAATAACGAGTGAATTTAGAGAAACAGAAAACGTGAAGATAACAGAGGAATCTAAGGATAGAGAAGGTAACAAAGTATCAGGTCCATATGAAAACTCAGAAAATTCCAATGTAACAAGTGAATCTGAAGAGACCAAAAAATTAGCCGAAAAAGAGGAGAATGAGGGAGAAAAATTAGGAGAAAATGTTAATGATGGGGCATCAGAAAATTCAGAAGATCCCAAAAAATTAACAGAACAAGAAGAAAATGGTACAAAGGAAAGTTCTGAAGAAACAAAAGATGATAAACCGGAAGAAAATGAGAAAAAGGCAGATAATAAAAAAAAAAGTAAAAAAAAGAAAAATCATTTTTTCAAATGTTAGGATGTAATTTCCTATGTAATAAAAATATTGAAACTGATGATGAAGAAGAAACGTTGGTAGTAAAAGATGATGCTAAAAAGAAACATAAATTTTTAAGAGAAGCTAATACTGAAAAAAATGATAATGAAAAGAAAGATAAATTATTAGGAGAAGGAGATAAAGAAGATGTTAAAGAAAAGAATGATGAACAGAAAGATAAAGTATTAGGAGAAGGAGATAAAGAAGATGTTAAAGAAAAGAATGATGAACAGAAAGATAAAGTATTAGGAGAAGGAGATAAAGAAGATGTTAAAGAAAAGAATGATGGAAAGAAAGATAAAGTGATAGGATCAGAAAAAACACAAAAGGAAATTAAAGAAAAAGTAGAAAAAAGAGTTAAAAAAAAGTGTAAAAAAAAAGTAAAAAAAGGAATTAAAGAAAATGATACTGAAGGTAACGATAAAGTGAAAGGACCAGAAATAATAATTGAAGAAGTAAAAGAAGAAATTAAAAAACAAGTAGAAGATGGAATTAAAGAAAATGATACTGAAGGTAACGATAAAGTGAAAGGGCCAGAAATAATAACTGAAGAAGTAAAAGAAGAAATTAAAAAACAAGTAGAAGAAGGAATTAAAGAAAATGATACTGAAGGTAACGATAAAGTGAAAGGGCCAGAAATAATAACTGAAGAAGTAAAAGAAGAAATTAAAAAACAAGTAGAAGAAGGAATTAAAGAAAATGATACTGAAAGTAAGGATAAATTGATAGGACAAGAAATAATAACTGAAGAA GTAAAAGAAGGAATTAAAGAAAATGATACTGAAAATAAAGATAAAGTGATAGGACAAGAAATAATAACTGAAGAAGTAAAAGAAGGAATTAAAGAAAATGATACTGAAAATAAAGATAAAGTGATAGGACAAGAAATAATAACTGAAGAAGTAAAAAAAGAAATTGAAAAACAAGAAGAAAAAGGAAATAAAGAAAATATTCTTGAAATTAAAGATATAGTAATTGGACAAGAAGTAATAATAGAAGAAGTAAAAAAAGTAATTAAAAAAAAAGTAGAAAAAGGAATTAAAGAAAATCATACTGAAAGTAAAGATAAAGTGATAGGACAAGAAATAATAGTTGAAGAAGTAAAAGAAGAAATTGAAAAACAAGTAGAAGAAGGAATTAAAGAAAATGATACTGAAAGTAAAGATAAAGTGATAGGACAAGAAGTGATAAAAGGAGATGTTAATGAAGAA GGTCCCGAAAACAAAGATAAAGTGACAAAACAGGAAAAAGTAAAAGAAGTTAAAAAAGAAGTAAAAAAAAAAGTTAAAAAAAGAGTAAAAAAAAGAAATAATAAGAATGAAAGAAAAGATAATGTGATAGGAAAAGAAATAATGAAAGAAGATGTTAATGAAAAAGATACCGCAAACAAAGATAAAGAGATAGAACAAGAAAAAGAAAAAGAAGAAGTTAAAGAAAAAGAAGAAGTTAAAGAAAAAGAAGAAGTTAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGATACCGAAAGCAAAGATAAAGAGATAGAACAAGAAAAAGAAAAAGAAGAAGTAAAAGAAGTTAAAGAAAAAGATACCGAAAACAAAGATAAAGTGATAGGACAAGAAATAATAATAGAAGAAATAAAAAAAGAAGTTAAAAAAAGAGTAAAAAAAAGAAATAATAAAAATGAAAACAAAGATAATGTGATAGTACAAGAAATAATGAACGAAGATGTTAACGAAAAAGATACCGCAAACAAAGATAAGGTGATAGAACAAGAAAAAGAAAAAGAAGAAGTTAAAGAAAAAGAAGAAGTTAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGAAGAAGTAAAAGAAAAAGATACCGAAAGCAAAGATAATGTGATAGTACAAGAAATAATGAACGAAGATGTTAACGAAAAAGATACCGAAAGCAAAGATAAAATGATAGGAAAAGAAGTAATAATAGAAGAAGTAAAAGAAGAAGTTAAAAAAAGAGTAAACAAAGAAGTTAACAAAAGAGTAAACAGAAGAAATAGAAAAAATGAAAGAAAAGATGTGATAGAACAAGAAATAGTAAGCGAAGAAGTTAACGAAAAAGATACCAAAAACAACGATAAAAAGATAGGAAAAAGAGTCAAAAAACCAATAGATGATTGTAAAAAAGAAAGAGAAGTACAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAGTCTGAAGAAGAGTCTGAAGAAGAGTCTGAAGAAGAATCTGAAGAAGAATCTGATGAAGAAAAAAATACATCAGGTTTGGTACATAGAAGAAATTGTAAAAAAGAAAAGAAATATAATAATGGAGAATTAGAAGAATATTATAAAGAGAAACAGAATGAAGAATATTTTGATGAAGAATATATTATTCAATCAAAAGAACATAATACTTTGAATACATTCCCAAATATGGCATTAAATGAAGATTTCAGAAGAGAATTTCACAATATATTAAGTATTCATGAAGATACAGATTTGATGGAACTAAAAAGAATCTTATATAATTTATTTTTAGAATATAATCCACATATGAATAATAAACAGAAAGCAGAATTGGATAAGAAATTTAGTGAAATGAATGTGGTACATCAAATATTAAATTATGAAGAGAGAATACGCATGTATGAAGAAAATGCAGCACGAGGAAGACTAAATACAGTTATTCTGGATCCAATTATTACATTTAATGTAATATTCGGAGATGATACAATGTTTAAGTTTATTGATGAATAA  Sequence Length: 4305 bp Clone #TL22: Plasmodium falciparum glutamic acid-rich protein (Pf-GARP)Nucleic acid sequence of Clone#TL22, 792 bp (Sequence1,231-2,022 of gene PFA_0620c) (SEQ ID NO: 25)TCAAAAGAACACAAATCAAAAGGAAAGAAAGATAAAGGAAAGAAAGATAAAGGAAAACATAAAAAAGCAAAAAAAGAAAAAGTAAAAAAACACGTAGTTAAAAATGTTATAGAAGATGAAGACAAAGATGGTGTAGAAATAATAAACTTAGAAGATAAAGAGGCATGTGAAGAACAACACATAACAGTAGAAAGTAGACCACTAAGCCAACCACAATGTAAACTAATAGATGAACCAGAACAATTAACATTAATGGATAAATCAAAAGTTGAAGAAAAAAACTTATCCATACAAGAGCAATTAATAGGTACCATAGGACGTGTTAATGTAGTACCCAGAAGAGATAATCATAAGAAAAAAATGGCGAAGATAGAGGAAGCTGAACTTCAAAAACAGAAACATGTTGATAAGGAAGAAGACAAAAAAGAAGAATCCAAAGAAGTAGAAGAAGAATCTAAAGAGGTACAAGAAGATGAAGAAGAAGTAGAAGAAGATGAAGAAGAAGAAGAAGAAGAAGAGGAAGAAGAAGAAGAAGAAGAAGAAGAAGAGGAAGAAGAAGAAGATGAAGTAGAAGAAGATGAAGATGATGCTGAAGAAGATGAAGATGATGCTGAAGAAGATGAAGATGATGCTGAAGAAGATGATGATGATGCTGAAGAAGATGATGATGATGCTGAAGAAGATGATGATGAAGATGAAGATGAAGATGAAGAAGAAGAAGAAGATGAAGAAGAAGAAGAAGAATCAGAAAAAAAAATAAAAAGAAATTTGAGAAAAAATGCCAAAATTTAA  Sequence Length: 792 Amino acid sequence of Clone#TL22 (SEQ ID NO: 26)SKEHKSKGKKDKGKKDKGKHKKAKKEKVKKHVVKNVIEDEDKDGVEIINLEDKEACEEQHITVESRPLSQPQCKLIDEPEQLTLMDKSKVEEKNLSIQEQLIGTIGRVNVVPRRDNHKKKMAKIEEAELQKQKHVDKEEDKKEESKEVEEESKEVQEDEEEVEEDEEEEEEEEEEEEEEEEEEEEEEDEVEEDEDDAEEDEDDAEEDEDDAEEDDDDAEEDDDDAEEDDDEDEDEDEEEEEDEEEEEESEKKIKRNLRKNAKI  Sequence Length: 263 Amino acid sequence of Pf-GARP (PFA_0620c) (SEQ ID NO: 27)MNVLFLSYNICILFFVVCTLNFSTKCFSNGLLKNQNILNKSFDSITGRLLNETELEKNKDDNSKSETLLKEEKDEKDDVPTTSNDNLKNAHNNNEISSSTDPTNIINVNDKDNENSVDKKKDKKEKKHKKDKKEKKEKKDKKEKKDKKEKKHKKEKKHKKDKKKEENSEVMSLYKTGQHKPKNATEHGEENLYEEMVSEINNNAQGGLLLSSPYQYREQGGCGIISSVHETSNDTKDNDKENISEDKKEDHQQEEMLKTLDKKERKQKEKEMKEQEKIEKKKKKQEEKEKKKQEKERKKQEKKERKQKEKEMKKQKKIEKERKKKEEKEKKKKKHDKENEETMQQPDQTSEETNNEIMVPLPSPLTDVTTPEEHKEGEHKEEEHKEGEHKEGEHKEEEHKEEEHKK EEHKSKEHKSKGKKDKGKKDKGKHKKAKKEKVKKHVVKNVIEDEDKDGVE II NLEDKEACEEQHITVESRPLSQPQCKLIDEPEQLTLMDKSKVEEKNLSIQEQLIGTIGRVNVVPRRDNHKKKMAKIEEAELQKQKHVDKEEDKKEESKEVEEESKEVQEDEEEVEEDEEEEEEEEEEEEEEEEEEEEEEDEVEEDEDDAEEDEDDAEEDEDDAEEDDDDAEEDDDDAEEDDDEDEDEDEEEEEDEEEEEESEKKIKRNLRKNAKI Sequence Length: 673 aa Coding Nucleic acid sequence gene Pf-GARP (PFA_0620c) (SEQ ID NO: 28)ATGAATGTGCTATTTCTTTCGTATAATATTTGTATTCTTTTTTTTGTTGTATGCACATTAAATTTTTCTACTAAGTGCTTTTCCAATGGTTTATTGAAGAATCAAAATATCCTAAACAAAAGTTTTGATTCCATAACGGGAAGATTATTAAACGAAACCGAATTAGAAAAAAATAAAGATGATAATTCAAAATCTGAAACGTTGTTAAAAGAGGAAAAAGATGAAAAGGATGATGTACCTACAACGAGTAATGACAACCTTAAGAATGCTCATAATAATAATGAAATTTCAAGTTCAACTGATCCAACGAATATTATTAATGTTAATGATAAAGATAATGAAAACTCTGTAGATAAAAAAAAAGATAAAAAAGAAAAAAAGCATAAAAAAGATAAAAAAGAAAAAAAAGAAAAAAAAGATAAAAAAGAAAAAAAAGATAAAAAAGAAAAAAAACATAAAAAAGAAAAAAAACATAAAAAAGATAAAAAAAAAGAAGAAAACAGTGAAGTGATGTCTTTATATAAAACGGGTCAACATAAACCAAAAAACGCAACAGAACATGGTGAAGAAAATTTATATGAAGAAATGGTAAGTGAAATAAATAATAATGCACAAGGTGGACTCCTTTTATCAAGCCCATATCAATATAGAGAACAAGGAGGATGTGGAATCATATCTAGTGTTCATGAGACGTCTAATGATACAAAAGATAATGATAAAGAAAATATATCCGAAGACAAAAAGGAGGACCATCAACAAGAAGAAATGTTGAAAACACTTGATAAAAAAGAACGTAAACAAAAAGAAAAAGAAATGAAAGAACAAGAAAAAATCGAAAAAAAAAAAAAAAAGCAAGAAGAAAAGGAAAAGAAAAAACAAGAAAAAGAAAGAAAAAAACAAGAAAAGAAAGAACGTAAACAAAAAGAAAAAGAAATGAAAAAACAAAAAAAAATAGAAAAAGAAAGAAAAAAGAAAGAAGAAAAGGAAAAGAAAAAGAAAAAACATGATAAGGAAAATGAAGAAACAATGCAACAACCAGATCAAACAAGTGAAGAAACCAACAATGAAATTATGGTACCATTACCAAGTCCATTGACAGACGTAACTACACCAGAAGAACACAAAGAAGGAGAACACAAAGAAGAAGAACACAAAGAAGGAGAACACAAAGAAGGAGAACACAAAGAAGAAGAACACAAAGAAGAAGAACACAAAAAAG AAGAACACAAATCAAAAGAACACAAATCAAAAGGAAAGAAAGATAAAGGAAAGAAAGATAAAGGAAAACATAAAAAAGCAAAAAAAGAAAAAGTAAAAAAACACGTAGTTAAAAATGTTATAGAAGATGAAGACAAAGATGGTGTAGAAATAATAAACTTAGAAGATAAAGAGGCATGTGAAGAACAACACATAACAGTAGAAAGTAGACCACTAAGCCAACCACAATGTAAACTAATAGATGAACCAGAACAATTAACATTAATGGATAAATCAAAAGTTGAAGAAAAAAACTTATCCATACAAGAGCAATTAATAGGTACCATAGGACGTGTTAATGTAGTACCCAGAAGAGATAATCATAAGAAAAAAATGGCGAAGATAGAGGAAGCTGAACTTCAAAAACAGAAACATGTTGATAAGGAAGAAGACAAAAAAGAAGAATCCAAAGAAGTAGAAGAAGAATCTAAAGAGGTACAAGAAGATGAAGAAGAAGTAGAAGAAGATGAAGAAGAAGAAGAAGAAGAAGAGGAAGAAGAAGAAGAAGAAGAAGAAGAAGAGGAAGAAGAAGAAGATGAAGTAGAAGAAGATGAAGATGATGCTGAAGAAGATGAAGATGATGCTGAAGAAGATGAAGATGATGCTGAAGAAGATGATGATGATGCTGAAGAAGATGATGATGATGCTGAAGAAGATGATGATGAAGATGAAGATGAAGATGAAGAAGAAGAAGAAGATGAAGAAGAAGAAGAAGAATCAGAAAAAAAAATAAAAAGAAATTTGAGAAAAAATGCCAAAATTTAA    Sequence Length: 2022 bpClone #TL27: Plasmodium falciparum 3D7 Plasmodium exported protein (PHISTc), unknown function (PFI1780w) mRNA, complete cdsNucleic acid sequence of Clone#TL27, 303 bp (Sequence691-998 of gene (PFI1780w) (SEQ ID NO: 29)GAACATGGTGAAATGCTAAATCAAAAAAGAAAACTTAAACAACATGAACTTGATAGAAGAGCACAAAGGGAAAAAATGTTAGAAGAACATAGTAGAGGAATATTTGCTAAAGGATATTTGGGAGAAGTAGAATCAGAAACTATAAAAAAGAAAACGGAACACCATGAAAATGTAAATGAAGATAATGTAGAAAAACCAAAATTGCAACAACATAAAGTTCAACCACCAAAAGTCCAACAACAAAAAGTTCAACCACCAAAATCACAACAACAAAAAGTTCAACCACCAAAATCACAACAACAA  Sequence Length: 303Amino acid sequence of Clone#TL27 (SEQ ID NO: 30)EHGEMLNQKRKLKQHELDRRAQREKMLEEHSRGIFAKGYLGEVESETIKKKTEHHENVNEDNVEKPKLQQHKVQPPKVQQQKVQPPKSQQQKVQPPKSQQQ  Sequence Length: 101Amino acid sequence of PFI1780w (SEQ ID NO: 31)MAVSTYNNTRRNGLRYVLKRRTILSVFAVICMLSLNLSIFENNNNNYGFHCNKRHFKSLAEASPEEHNNLRSHSTSDPKKNEEKSLSDEINKCDMKKYTAEEINEMINSSNEFINRNDMNIIFSYVHESEREKFKKVEENIFKFIQSIVETYKIPDEYKMRKFKFAHFEMQGYALKQEKFLLEYAFLSLNGKLCERKKFKEVLEYVKREWIEFRKSMFDVWKEKLASEFREHGEMLNQKRKLKQHELDRRAQREKMLEEHSRGIFAKGYLGEVESETIKKKTEHHENVNEDNVEKPKLQQHKVQPPKVQQQKVQPPKSQQQKVQPPKSQQQKVQPPKVQQQKVQPPKVQKPKLQNQKGQKQVSPKAKGNNQAKPTKGNKLKKN  Sequence Length: 383 aaCoding Nucleic acid sequence gene PFI1780w (SEQ ID NO: 32)ATGGCTGTTAGTACATATAATAATACTCGAAGGAATGGTCTAAGATATGTCCTTAAAAGACGTACCATTCTATCTGTTTTTGCTGTCATTTGTATGTTATCATTGAATTTATCAATATTTGAAAATAATAATAATAATTATGGATTCCATTGCAATAAAAGACATTTTAAAAGTTTAGCTGAAGCAAGTCCAGAAGAACATAACAATTTAAGAAGTCATTCAACAAGTGATCCAAAGAAGAATGAAGAGAAATCATTAAGTGACGAAATAAATAAATGTGATATGAAAAAATACACTGCTGAAGAAATAAATGAAATGATTAACAGTTCTAATGAATTTATAAATAGAAATGATATGAATATAATATTTAGTTATGTACATGAATCTGAGAGAGAAAAATTTAAAAAGGTAGAAGAAAATATATTTAAATTTATTCAAAGTATAGTAGAAACATATAAAATACCAGATGAATATAAAATGAGAAAATTCAAATTTGCACACTTTGAAATGCAAGGATATGCATTAAAACAAGAAAAGTTCCTTTTAGAATATGCTTTTCTTTCCTTAAATGGTAAATTATGTGAACGTAAAAAATTTAAAGAAGTTTTAGAATATGTAAAAAGGGAATGGATTGAGTTTAGAAAATCAATGTTTGACGTATGGAAGGAAAAATTAGCTTCTGAATTCAGAGAACATGGTGAAATGCTAAATCAAAAAAGAAAACTTAAACAACATGAACTTGATAGAAGAGCACAAAGGGAAAAAATGTTAGAAGAACATAGTAGAGGAATATTTGCTAAAGGATATTTGGGAGAAGTAGAATCAGAAACTATAAAAAAGAAAACGGAACACCATGAAAATGTAAATGAAGATAATGTAGAAAAACCAAAATTGCAACAACATAAAGTTCAACCACCAAAAGTCCAACAACAAAAAGTTCAACCACCAAAATCACAACAACAAAAAGTTCAACCACCAAAATCACAACAACAAAAAGTTCAACCACCAAAAGTACAACAACAAAAAGTTCAACCACCAAAAGTGCAAAAACCAAAACTTCAAAATCAAAAAGGACAAAAGCAAGTATCTCCCAAAGCAAAGGGTAATAATCAAGCGAAACCAACCAAAGGAAACAAGTTAAAGAAAAATTAA Sequence Length: 1152 bp Clone #TL5: Plasmodium falciparum 3D7 knob-associatedhistidine-rich protein (PFB0100c)Nucleic acid sequence of Clone#TL5, 242 bp (Sequence1309-1550 of gene (PFB0100c) (SEQ ID NO: 33)GTTAAAGAAAAGGGAGAAAAGCATAATGGAAAAAAACCATGCAGCAAAAAAACTAACGAAGAAAATAAAAATAAAGAAAAAACCAATAATTCAAAATCAGATGGATCAAAAGCTCATGAAAAAAAAGAAAATGAAACAAAAAACACCGCTGGAGAAAATAAAAAAGTAGATTCTACTTCAGCTGATAATAAATCAACAAATGCTGCTACACCAGGCGCAAAAGATAAAACTCAAGGAGGAAA  Sequence Length: 242 bp Amino acid sequence of Clone#TL5 (SEQ ID NO: 34)VKEKGEKHNGKKPCSKKTNEENKNKEKTNNSKSDGSKAHEKKENETKNTAGENKKVDSTSADNKSTNAATPGAKDKTQGG  Sequence Length: 80 aa Amino acid sequence of PFB0100c (SEQ ID NO: 35)MKSFKNKNTLRRKKAFPVFTKILLVSFLVWVLKCSNNCNNGNGSGDSFDFRNKRTLAQKQHEHHHHHHHQHQHQHQAPHQAHHHHHHGEVNHQAPQVHQQVHGQDQAHHHHHHHHHQLQPQQPQGTVANPPSNEPVVKTQVFREARPGGGFKAYEEKYESKHYKLKENVVDGKKDCDEKYEAANYAFSEECPYTVNDYSQENGPNIFALRKRFPLGMNDEDEEGKEALAIKDKLPGGLDEYQNQLYGICNETCTTCGPAAIDYVPADAPNGYAYGGSAHDGSHGNLRGHDNKGSEGYGYEAPYNPGFNGAPGSNGMQNYVPPHGAGYSAPYGVPHGAAHGSRYSSFSSVNKYGKHGDEKHHSSKKHEGNDGEGEKKKKSKKHKDHDGEKKKSKKHKDNEDAESVKSKKHKSHDCEKKKSKKHKDNEDAESVKSKKS VKEKGEKHNGKKPCSKKTNEENKNKEKTNNSKSDGSKAHEKKENETKNTAGENKKVDSTSADNKSTNAATPGAKDKTQGG KTDKTGASTNAATNKGQCAAEGATKGATKEASTSKEATKEASTSKEATKEASTSKEATKEASTSKGATKEASTTEGATKGASTTAGSTTGATTGANAVQSKDETADKNAANNGEQVMSRGQAQLQEAGKKKKKRGCCG  Sequence Length: 654 aa Coding Nucleic acid sequence gene PFB0100c (SEQ ID NO: 36)ATGAAAAGTTTTAAGAACAAAAATACTTTGAGGAGAAAGAAGGCTTTCCCTGTTTTTACTAAAATTCTTTTAGTCTCTTTTTTAGTATGGGTTTTGAAGTGCTCTAATAACTGCAATAATGGAAACGGATCCGGTGACTCCTTCGATTTCAGAAATAAGAGAACTTTAGCACAAAAGCAACATGAACACCATCACCACCATCACCATCAACATCAACACCAACACCAAGCTCCACACCAAGCACACCACCATCATCATCATGGAGAAGTAAATCACCAAGCACCACAGGTTCACCAACAAGTACATGGTCAAGACCAAGCACACCATCACCATCATCACCACCATCATCAATTACAACCTCAACAACCCCAGGGAACAGTTGCTAATCCTCCTAGTAATGAACCAGTTGTAAAAACCCAAGTATTCAGGGAAGCAAGACCAGGTGGAGGTTTCAAAGCATATGAAGAAAAATACGAATCAAAACACTATAAATTAAAGGAAAATGTTGTCGATGGTAAAAAAGATTGTGATGAAAAATACGAAGCTGCCAATTATGCTTTCTCCGAAGAGTGCCCATACACCGTAAACGATTATAGCCAAGAAAATGGTCCAAATATATTTGCCTTAAGAAAAAGATTCCCTCTTGGAATGAATGATGAAGATGAAGAAGGTAAAGAAGCATTAGCAATAAAAGATAAATTACCAGGTGGTTTAGATGAATACCAAAACCAATTATATGGAATATGTAATGAGACATGTACCACATGTGGACCTGCCGCTATAGATTATGTTCCAGCAGATGCACCAAATGGCTATGCTTATGGAGGAAGTGCACACGATGGTTCTCACGGTAATTTAAGAGGACACGATAATAAAGGTTCAGAAGGTTATGGATATGAAGCTCCATATAACCCAGGATTTAATGGTGCTCCTGGAAGTAATGGTATGCAAAATTATGTCCCACCCCATGGTGCAGGCTATTCAGCTCCATACGGAGTTCCACATGGTGCAGCCCATGGTTCAAGATATAGTTCATTCAGTTCCGTAAATAAATATGGAAAACACGGTGATGAAAAACACCATTCCTCTAAAAAGCATGAAGGAAATGACGGTGAAGGAGAAAAAAAGAAAAAATCAAAAAAACACAAAGACCACGATGGAGAAAAGAAAAAATCAAAAAAACACAAAGACAATGAAGATGCAGAAAGCGTAAAATCAAAAAAACACAAAAGCCACGATTGTGAAAAGAAAAAATCAAAAAAACACAAAGACAATGAAGATGCAGAAAGCGT AAAATCAAAAAAAAGTGTTAAAGAAAAGGGAGAAAAGCATAATGGAAAAAAACCATGCAGCAAAAAAACTAACGAAGAAAATAAAAATAAAGAAAAAACCAATAATTCAAAATCAGATGGATCAAAAGCTCATGAAAAAAAAGAAAATGAAACAAAAAACACCGCTGGAGAAAATAAAAAAGTAGATTCTACTTCAGCTGATAATAAATCAACAAATGCTGCTACACCAGGCGCAAAAGATAAAACTCAAGGAGGAAA AACTGACAAAACAGGAGCAAGTACTAATGCCGCAACAAATAAAGGACAATGTGCTGCTGAAGGAGCAACTAAGGGAGCAACTAAAGAAGCAAGTACTTCTAAAGAAGCAACAAAAGAAGCAAGTACTTCTAAAGAAGCAACAAAAGAAGCAAGTACTTCTAAAGAAGCAACAAAAGAAGCAAGTACTTCTAAAGGAGCAACTAAAGAAGCAAGTACTACTGAAGGAGCAACTAAAGGAGCAAGTACTACTGCAGGTTCAACTACAGGAGCAACTACAGGAGCTAATGCAGTACAATCTAAAGATGAAACTGCCGATAAAAATGCTGCAAATAATGGTGAACAAGTAATGTCAAGAGGACAAGCACAATTACAAGAAGCAGGAAAGAAAAAGAAGAAAAGAGGATGCTGTGGTTAA Sequence Length: 1965 bp Clone #TL16: Plasmodium falciparum isolate 822 rhoptryassociated membrane antigen gene (MAL7P1.208)Nucleic acid sequence of Clone#TL16, 432 bp (Sequence953-,1384 of gene MAL7P1.208) (SEQ ID NO: 37)GAAGAATCCAAAAATGAAGAATTTAAAAATGAAGAATTCAAAAATGTAGATAAAGAAAATTATGATGATAAAAATATTTTCTATGGTTATAGTGATAATGATGATGAAAGCTTTTTAGAAACTGATTCTTATGAAGAATATGAAGACGAAGATAAAGATGTTGAAGATGAGTATGAAGAAAGTTTCTTACAAAATGATGAGAAAAAAATGGTCTTTTATGATTTATACAAGCCAGAAGAAAATGAATCTTATTATGAAAAGAAACAAAAGAAAGAAGAAAAAGAAGAGAAAGAAGAGAAAGAACAAAGTTTGAACAAACAAAACGATATGGAAGACCAAGAAGATAATGAAGAATATAAATTTGAAGAAGAAAATAAAGAAGACCTTCTAGATGTCCAACAAGATGAAGAATTACCAAGTGAAGGAAAACAA  Sequence Length: 432Amino acid sequence of Clone#TL16 (SEQ ID NO: 38)EESKNEEFKNEEFKNVDKENYDDKNIFYGYSDNDDESFLETDSYEEYEDEDKDVEDEYEESFLQNDEKKMVFYDLYKPEENESYYEKKQKKEEKEEKEEKEQSLNKQNDMEDQEDNEEYKFEEENKEDLLDVQQDEELPSEGKQ  Sequence Length: 144 Amino acid sequence of MAL7P1.208 (SEQ ID NO: 39)ISFSDYERSIKNFSISSHAENNYDNIINEYKKIKDINNNINILSSVHRKGRILYDSFLEINKLENDKKEKHEKEDEYEDNDESFLETEEYEDNEDEKYNKDEDDYAESFIETDEYEDNEDDKYNKDEDDYSESFIETDEYDDNEEEQYNKDEDDYADSFIETDHYENNDDKNEEEEEYNDQDNDYGYNFLETDEYDDSEEYDYDDKEYGESFLEKEEGEEMKDEEMKDEEMKDVEMKDEEMKDEEIKYDEMKNEEMKYDEMKDEVMKDEEMKDEVMKDEEMKDEQMKYEEF KNEESKNEESKNEESKNEESKNEEFKNEESKNEEFKNEEFKNVDKENYDDKNIFYGYSDNDDESFLETDSYEEYEDEDKDVEDEYEESFLQNDEKKMVFYDLYKPEENESYYEKKQKKEEKEEKEEKEQSLNKQNDMEDQEDNEEYKFEEENKEDLLDVQQDEELPSEGKQ KVKGKSFDNEHLNEIQNVSDVHAFIQKDMKYLDDLIDEEQTIKDAVKKSAYKGNKKLGNNKKSQMILEEEPEENFEEDADEELNKLMEQEKNIVDKEIKNSKANKSNKKLQFNNTNKQNKMYMKNEYNNKTKNNKNNKFEQQNYDESYMDDDYEQNEEFNDNNQSEDMKETNELDKINDELLTDQGPNEDTLLENNNKIFDNKFVAHKKREKSISPHSYQKVSTKVQNKEDMENKEEKQLISSequence Length: 704  Coding Nucleic acid sequence gene MAL7P1.208(SEQ ID NO: 40)ATTAGCTTTTCTGATTATGAGAGATCAATAAAAAACTTTTCTATTTCTTCTCATGCAGAAAATAATTATGATAATATAATAAATGAATATAAAAAAATAAAAGATATTAACAACAATATAAACATATTATCATCAGTACATAGAAAAGGAAGAATATTGTACGACAGCTTTTTAGAAATAAATAAGTTGGAAAATGACAAAAAAGAGAAACATGAAAAAGAAGATGAATATGAAGATAATGATGAAAGCTTTTTAGAAACTGAAGAATATGAAGATAATGAAGATGAAAAATATAACAAAGATGAAGATGATTATGCAGAAAGTTTTATTGAGACTGATGAATATGAAGATAATGAAGATGATAAATATAATAAAGATGAAGATGATTATTCAGAAAGCTTTATTGAGACTGATGAATATGATGATAATGAAGAAGAACAATATAATAAAGATGAAGATGATTATGCAGATAGTTTTATTGAGACAGACCATTATGAAAATAACGATGATAAAAATGAAGAAGAAGAAGAATATAATGATCAAGATAATGATTATGGATATAACTTTTTAGAAACTGACGAATACGATGATAGCGAAGAATATGATTACGACGATAAGGAATACGGAGAGAGTTTCCTCGAAAAAGAAGAAGGTGAAGAAATGAAAGATGAAGAGATGAAAGATGAAGAAATGAAAGATGTAGAAATGAAAGATGAAGAGATGAAAGATGAAGAGATAAAATATGACGAGATGAAAAATGAAGAGATGAAATATGACGAGATGAAAGATGAAGTGATGAAAGATGAAGAGATGAAAGATGAAGTGATGAAAGATGAAGAGATGAAAGACGAACAAATGAAATATGAAGAATTCAAAAAT GAAGAATCCAAAAATGAAGAATCCAAAAATGAAGAATCCAAAAATGAAGAATCCAAAAATGAAGAATTCAAAAATGAAGAATCCAAAAATGAAGAATTTAAAAATGAAGAATTCAAAAATGTAGATAAAGAAAATTATGATGATAAAAATATTTTCTATGGTTATAGTGATAATGATGATGAAAGCTTTTTAGAAACTGATTCTTATGAAGAATATGAAGACGAAGATAAAGATGTTGAAGATGAGTATGAAGAAAGTTTCTTACAAAATGATGAGAAAAAAATGGTCTTTTATGATTTATACAAGCCAGAAGAAAATGAATCTTATTATGAAAAGAAACAAAAGAAAGAAGAAAAAGAAGAGAAAGAAGAGAAAGAACAAAGTTTGAACAAACAAAACGATATGGAAGACCAAGAAGATAATGAAGAATATAAATTTGAAGAAGAAAATAAAGAAGACCTTCTAGATGTCCAACAAGATGAAGAATTACCAAGTGAAGGAAAACAAAAAGTAAAAGGAAAATCATTCGATAATGAACATTTGAATGAAATACAAAATGTTAGCGACGTACATGCATTTATACAAAAAGATATGAAATATTTAGATGATCTCATAGATGAAGAGCAAACTATTAAAGATGCCGTCAAAAAAAGTGCTTATAAAGGAAATAAGAAATTAGGAAATAATAAAAAATCACAAATGATACTGGAAGAAGAACCAGAAGAAAATTTTGAAGAAGATGCTGATGAAGAATTAAATAAACTAATGGAACAAGAAAAAAATATTGTAGATAAAGAAATCAAAAATAGTAAAGCAAATAAAAGCAACAAAAAATTACAATTCAATAACACTAATAAACAAAACAAAATGTATATGAAAAACGAATATAATAATAAGACAAAAAATAATAAAAACAA TAAATTTGAACAACAAAATTATGATGAATCATATATGGATGATGATTATGAACAAAATGAAGAATTTAATGATAATAATCAAAGCGAAGATATGAAAGAAACAAATGAACTCGATAAAATTAATGATGAACTATTAACTGATCAAGGACCAAACGAAGATACATTATTAGAAAATAATAATAAAATTTTCGATAATAAATTTGTAGCACATAAAAAAAGAGAAAAAAGTATATCCCCACACAGTTACCAAAAGGTATCTACCAAAGTACAAAATAAGGAAGACATGGAAAATAAGGAAGAGAAACAA TTGATAAGTAA Sequence Length: 2114 Clone #TL45: Plasmodium falciparum 3D7 Cg4 protein (PF07_0033)Nucleic acid sequence of Clone#TL45, 650 bp (Sequence 1,764-2413 of gene PF07_0033) (SEQ ID NO: 41)TCACCAAATAAAACAGAATTAAAAAAAGGAGAAGAAGGAAAAGTACAAACATGTTATACAACAATACCTATTGAAACATTATTAGCTCAAGGATCTTATAGTTCTAAAGATATATTCAATTTTAGTGAACAGGAAATTAATATGCAACATAGTGATATATTAGAAGGAGAACGATTAAAACATCTTAATGAACTAGAAACTATTATATATGAAAGTAGAAGTAGACTTAATGGTATATATAAAAATTTTGTTATGGATGATGAAAGAGATCGTATTTTACTTTCCTTAGATGATTATGAAAATTGGTTATATGATAATATAGAAGAAAATAAAAATATGTTTATTAAAAAAAAAGAAGAAATTAGAGATCTTATAAAAAATATTGTACAAAAATTTGATGTATATAATTCAAAACAACAAAATCTAGGAAATATAATTAATCATCTTAATAATATCATAACACAATGTTCAAATAAACCATCGGATGAAAGTCAAAATATAATTAATAGAACAACGAAATTCTTAAATAATATTAATTCTTTACAAGAACAAGAAAAAAATAAACCACTATACGAACCACCTGTATATACACTTAACGATATTGAAGCAGAATTTAATGAAGTCACACAACTCGCTCAAAAATTCTTTTC  Sequence Length: 650 bpAmino acid sequence of Clone#TL45 (SEQ ID NO: 42)SPNKTELKKGEEGKVQTCYTTIPIETLLAQGSYSSKDIFNFSEQEINMQHSDILEGERLKHLNELETIIYESRSRLNGIYKNFVMDDERDRILLSLDDYENWLYDNIEENKNMFIKKKEEIRDLIKNIVQKFDVYNSKQQNLGNIINHLNNIITQCSNKPSDESQNIINRTTKFLNNINSLQEQEKNKPLYEPPVYTLNDIEAEFNEVTQLAQKFF  Sequence Length: 216 aaAmino acid sequence of gene PF07_0033 (SEQ ID NO: 43)MSVLGIDIGNDNSVVATINKGAINVVRNDISERLTPTLVGFTEKERLIGDSALSKLKSNYKNTCRNIKNLIGKIGTDVKDDIEIHEAYGDLIPCEYNYLGYEVEYKNEKVVFSAVRVLSALLSHLIKMAEKYIGKECKEIVLSYPPTFTNCQKECLLAATKIINANVLRIISDNTAVALDYGMYRMKEFKEDNGSLLVFVNIGYANTCVCVARFFSNKCEILCDIADSNLGGRNLDNELIKYITNIFVNNYKMNPLYKNNTPELCPMGTGRLNKFLVTSTASDQQNGINNKVRIKLQEVAIKTKKVLSANNEASIHVECLYEDLDCQGSINRETFEELCSNFFLTKLKHLLDTALCISKVNIQDIHSIEVLGGSTRVPFIQNFLQQYFQKPLSKTLIADESIARGCVLSAAMVSKHYKVKEYECVEKVTHPINVEWHNINDASKSNVEKLYTRDSLKKKVKKIVIPEKGHIKLTAYYENTPDLPSNCIKELGSCIVKINEKNDKIVESHVMTTFSNYDTFTFLGAQTVTKSVIKSKDEKKKADDKTEDKGEKKDAKDQEQNDDKDQTNDNNMNEKDTNDKKEKNNETNSPNKTELKKGEEGKVQTCYTTIPIETLLAQGSYSSKDIFNFSEQEINMQHSDILEGERLKHLNELETIIYESRSRLNGIYKNFVMDDERDRILLSLDDYENWLYDNIEENKNMFIKKKEEIRDLIKNIVQKFDVYNSKQQNLGNIINHLNNIITQCSNKPSDESQNIINRTTKFLNNINSLQEQEKNKPLYEPPVYTLNDIEAEFNEVTQLAQKFFSKLEVEELAKQKAKQEKEKEKEKEKEKEKEKEKNEETNLDANEEQNNEAKNNEEKENSTKNENSANPEE  Sequence Length: 873 aaCoding Nucleic acid sequence gene PF07_0033 (SEQ ID NO: 44)ATGTCGGTTTTAGGTATAGATATAGGAAATGACAATTCTGTTGTAGCTACTATTAATAAAGGTGCTATAAATGTTGTGAGGAATGACATATCCGAAAGGTTAACCCCGACATTAGTTGGTTTCACCGAAAAAGAAAGATTAATAGGTGATAGTGCTTTATCTAAATTGAAATCTAATTATAAGAATACATGTAGGAATATAAAGAATTTGATAGGTAAAATAGGTACCGATGTAAAAGATGATATAGAAATACATGAAGCATATGGGGATTTAATACCATGTGAATATAATTATTTAGGTTATGAAGTTGAATATAAAAATGAAAAAGTTGTATTTAGTGCTGTTCGTGTTTTATCAGCCTTATTATCACATTTGATTAAAATGGCTGAAAAATATATTGGAAAGGAATGTAAAGAAATTGTCTTATCATATCCTCCAACATTTACAAATTGTCAAAAAGAATGTTTATTAGCTGCAACTAAAATTATTAATGCTAATGTTTTGAGAATTATTAGTGATAATACAGCTGTTGCTCTAGATTATGGAATGTACAGAATGAAAGAATTCAAAGAAGATAATGGATCCTTACTAGTTTTTGTTAACATTGGTTATGCAAATACTTGTGTATGTGTTGCGCGTTTTTTTTCTAATAAATGTGAAATCTTATGTGATATTGCTGATTCAAATTTAGGTGGTAGAAATTTAGATAATGAACTTATTAAATATATTACAAATATATTTGTTAATAATTATAAAATGAATCCATTATATAAAAACAATACTCCGGAATTATGCCCCATGGGTACTGGTAGATTAAATAAGTTTTTAGTAACATCTACAGCATCTGATCAACAAAATGGTATTAATAATAAAGTACGTATTAAATTACAAGAAGTTGCTATAAAAACAAAGAAAGTACTTTCAGCAAATAATGAAGCGTCCATACATGTTGAATGTTTATATGAAGATTTAGATTGTCAAGGTTCCATTAATAGAGAAACCTTTGAAGAATTGTGTTCAAACTTCTTCTTAACAAAATTAAAACATCTTCTAGATACTGCTCTATGTATTAGTAAAGTAAACATACAAGATATACATTCTATTGAAGTTTTGGGTGGATCCACAAGAGTTCCATTTATTCAAAATTTTTTACAACAATATTTTCAGAAACCATTATCTAAGACCCTTATAGCAGATGAATCTATAGCAAGAGGTTGTGTACTATCAGCTGCTATGGTTAGTAAACATTATAAAGTAAAAGAATATGAATGTGTAGAAAAAGTTACACATCCAATTAATGTTGAATGGCATAATATTAATGACGCATCTAAAAGTAATGTAGAAAAATTATATACAAGAGATTCCTTAAAAAAGAAAGTTAAGAAAATTGTTATCCCAGAAAAAGGACACATTAAACTTACAGCTTATTATGAAAATACACCAGATTTACCATCCAATTGTATAAAAGAATTGGGATCATGTATTGTTAAAATAAATGAAAAGAATGATAAAATTGTTGAATCCCACGTTATGACCACCTTTTCAAATTATGATACATTTACATTTTTAGGTGCACAGACAGTAACCAAGTCTGTTATTAAGTCCAAGGATGAAAAAAAAAAAGCAGATGACAAAACGGAGGATAAGGGAGAAAAAAAAGATGCAAAAGATCAAGAACAAAATGATGATAAAGATCAAACAAATGATAATAACATGAATGAGAAAGATACTAATGATAAAAAAGAAAAAAATAATGAAACAAACTCACCAAATAAAACAGAATTAAAAAAAGGAGAAGAAGGAAAAGTACAAACATGTTATACAACAATACCTATTGAAACATTATTAGCTCAAGGATCTTATAGTTCTAAAGATATATTCAATTTTAGTGAACAGGAAATTAATATGCAACATAGTGATATATTAGAAGGAGAACGATTAAAACATCTTAATGAACTAGAAACTATTATATATGAAAGTAGAAGTAGACTTAATGGTATATATAAAAATTTTGTTATGGATGATGAAAGAGATCGTATTTTACTTTCCTTAGATGATTATGAAAATTGGTTATATGATAATATAGAAGAAAATAAAAATATGTTTATTAAAAAAAAAGAAGAAATTAGAGATCTTATAAAAAATATTGTACAAAAATTTGATGTATATAATTCAAAACAACAAAATCTAGGAAATATAATTAATCATCTTAATAATATCATAACACAATGTTCAAATAAACCATCGGATGAAAGTCAAAATATAATTAATAGAACAACGAAATTCTTAAATAATATTAATTCTTTACAAGAACAAGAAAAAAATAAACCACTATACGAACCACCTGTATATACACTTAACGATATTGAAGCAGAATTTAATGAAGTCACACAACTCGCTCAAAAATTCTTTTCAAAGCTTGAAGTAGAAGAACTAGCCAAACAAAAAGCAAAGCAAGAAAAGGAAAAGGAAAAGGAAAAAGAAAAAGAGAAAGAAAAAGAAAAGGAAAAAAATGAAGAGACAAACTTGGATGCAAATGAGGAACAAAATAATGAAGCAAAAAATAATGAAGAAAAGGAGAACTCAACAAAAAATGAAAATTCAGCTAATCCAGAGGAATAA  Sequence Length: 2622 bpPlasmodium falciparum calcium-dependent protein kinase(PF-CDPK5), putative Gene PF3D7_1337800 (fragment C)Nucleic acid sequence 255 bp (Sequence 1452-1706(255) of gene PF3D7_1337800(SEQ ID NO: 45)TTCTTAGCAGCTTGTTTAGATCATAGTATATTTCAACAAGATGTTATCTGTAGAAATGCTTTCAATGTTTTTGATTTAGATGGTGATGGTGTTATAACAAAGGATGAATTATTTAAAATTCTATCCTTTAGTGCTGTACAAGTATCCTTTAGTAAAGAAATTATTGAAAATCTTATTAAAGAAGTCGATTCTAATAATGATGGATTTATAGATTATGATGAATTTTATAAGATGATGACGGGAGTTAAAGAATGASequence Length: 255  Amino acid sequence of Fragment C (Pf-CDPK5)(SEQ ID NO: 46)FLAACLDHSIFQQDVICRNAFNVFDLDGDGVITKDELFKILSFSAVQVSFSKEIIENLIKEVDSNNDGFIDYDEFYKMMTGVKE Sequence Length: 84 Amino acid sequence of PF3D7_1337800(Pf-CDPK5) (SEQ ID NO: 47)MKETEVEDMDTNRKDGKIKKKEKIVNMKNEEVKSTTKSTLADSDEDYSIITLCTKCLSKKLEDNKNRIILDSKAFKDNRLKGRCSVSSNEDPLDNKLNLSPYFDRSQIIQEIILMNNDELSDVYEIDRYKLGKGSYGNVVKAVSKRTGQQRAIKIIEKKKIHNIERLKREILIMKQMDHPNIIKLYEVYEDNEKLYLVLELCDGGELFDKIVKYGSFSEYEAYKIMKQIFSALYYCHSKNIMHRDLKPENILYVDNTEDSPIQIIDWGFASKCMNNHNLKSVVGTPYYIAPEILRGKYDKRCDIWSSGVIMYILLCGYPPFNGKNNDEILKKVEKGEFVFDSNYWARVSDDAKDLICQCLNYNYKERIDVEQVLKHRWFKKFKSNNLIINKTLNKTLIEKFKEFHKLCKIKKLAVTCIAYQLNEKDIGKLKKTFEAFDHNGDGVLTISEIFQCLKVNDNEFDRELYFLLKQLDTDGNGLIDYTEFLAACLDHSIFQQDVICRNAFNVFDLDGDGVITKDELFKILSFSAVQVSFSKEIIENLIKEVDSNNDGFIDYDEFYKMMTGVKE Sequence Length: 568 aa Coding Nucleotide sequence of PF3D7_1337800 (Pf-CDPK5) (SEQ ID NO: 48)ATGAAAGAGACGGAGGTCGAAGATATGGATACGAATAGAAAAGATGGTAAAATTAAAAAGAAAGAAAAAATAGTAAATATGAAAAATGAAGAAGTGAAAAGTACGACAAAGAGTACGTTAGCCGATAGTGATGAAGACTATTCGATTATAACTTTATGTACGAAATGTTTATCTAAAAAACTTGAAGATAATAAGAATCGAATAATTCTTGATAGTAAAGCTTTTAAAGATAATAGATTAAAAGGTAGATGTAGTGTTAGTTCCAATGAAGATCCTTTAGATAACAAATTAAATTTATCACCATATTTTGATAGATCCCAAATAATTCAAGAAATAATTTTGATGAATAATGATGAATTAAGTGATGTATATGAAATAGATAGATACAAGTTAGGCAAAGGATCTTATGGAAATGTTGTTAAAGCCGTAAGTAAAAGAACTGGTCAACAGAGAGCTATAAAAATTATAGAGAAAAAGAAAATTCATAATATTGAAAGATTAAAAAGAGAAATATTAATAATGAAACAGATGGATCATCCTAATATTATAAAATTATATGAAGTTTATGAAGACAATGAAAAATTATATTTAGTATTAGAATTATGTGACGGTGGAGAATTATTTGATAAAATTGTAAAATATGGTAGCTTCTCTGAATATGAAGCATATAAAATTATGAAACAAATATTTTCAGCTTTATATTATTGTCATAGTAAAAATATTATGCATAGAGATTTAAAACCAGAAAATATTTTATATGTAGATAATACAGAAGATTCTCCTATACAAATAATTGATTGGGGATTCGCTAGTAAATGTATGAATAATCATAATTTGAAATCAGTTGTTGGGACACCTTATTATATAGCACCCGAAATATTAAGAGGTAAATATGACAAAAGATGTGATATATGGAGTAGTGGTGTAATTATGTATATTTTATTATGTGGATATCCACCATTTAATGGAAAAAATAATGATGAAATCTTAAAAAAAGTGGAAAAAGGAGAATTTGTTTTCGATTCCAATTATTGGGCAAGAGTTAGTGATGATGCTAAAGATTTAATTTGTCAATGTTTAAATTATAATTATAAAGAAAGAATAGATGTTGAGCAAGTTCTAAAACATAGATGGTTCAAAAAATTTAAATCAAATAATCTTATTATAAATAAAACATTAAATAAAACTTTAATCGAAAAATTTAAAGAATTCCATAAATTATGTAAAATTAAAAAGCTAGCTGTAACATGTATAGCATACCAATTAAATGAAAAAGATATAGGGAAATTAAAAAAAACATTTGAAGCTTTTGATCATAATGGAGATGGAGTATTAACCATATCAGAAATTTTTCAATGTTTAAAAGTTAATGACAATGAATTTGATAGAGAATTATACTTTTTATTAAAACAACTTGATACAGATGGAAATGGATTAATTGATTATACTGAATTCTTAGCAGCTTGTTTAGATCATAGTATATTTCAACAAGATGTTATCTGTAGAAATGCTTTCAATGTTTTTGATTTAGATGGTGATGGTGTTATAACAAAGGATGAATTATTTAAAATTCTATCCTTTAGTGCTGTACAAGTATCCTTTAGTAAAGAAATTATTGAAAATCTTATTAAAGAAGTCGATTCTAATAATGATGGATTTATAGATTATGATGAATTTTATAAGATGATGACGGGAGTTAAAGAATGA Sequence Length: 1707 bp PbSEP-1; Gene PBANKA_050600 (PbSEP-1A)Nucleic acid sequence of PB Clone #2 828 bp (Sequence 2172-2991of gene PBANKA_050600) (SEQ ID NO: 65)TTAAAAGATAGTGATGGATATGAGAAATTATTAAAAAATGACATGTACGATTTATATAATATTAAGATGCATGATTTAAATAACTTAAAATCATATGATTTTGAATTTTCAAAAAATTTATTAAAAAACGAGATTTTTTTTTGTGGTGATAATATAAAAAGTGATGAAATAAATTTAAATGATAATGACATAAATGAAAAGATTGATTCACTAATGAACAATTACAATATTATGAAAAACAAACGTGACAAATTTAATGAAGAAGAAAACGAAATTCAAAACTTTTTAGCAGAATTAAAAGCTGATGTAACTAATCAACTCAATCTAAATAACGGGGAAGATGAACAGGCTTTTGATTTGCTTAATTCGTTTGATATAAACAATAACTTTGACGATTTTGTTGGCAACTTTGATGATACAAATGATAACATAGCTCAAAATAAATCAGACATAGACAATAATAAAGAGTTCGAACACGAAAATGATATAAATCATGATTATAACGATTGTGGTACATATATGGATGATATATATAATAACAATAATGGTGATGATATTTCGAGAAAGGGATCACGTCTGAAATTGTCTGATTTAAATGACGAAAAGAATTTATTTCCAGATGTCAACTCCTCTTTTAATACTCCTATAAAATCTTCTGAACTAAAGAGAGATTCAGAATGCCAAACAAATTCACCACTTATATTTTCTAGAAGTAATAGAACTCCTAGGAAAAAAAGTGTAGAAGTAATATTAGTAAAGAAAAAATTAAAAAAAAGAAAAGAAAAAGAATCAAATATATCATTTGAAAATACAACACATGATGATTATSequence Length: 828 bp  PBANKA_050600 (PbSEP-1A aa 724-997)(SEQ ID NO: 66)LKDSDGYEKLLKNDMYDLYNIKMHDLNNLKSYDFEFSKNLLKNEIFFCGDNIKSDEINLNDNDINEKIDSLMNNYNIMKNKRDKFNEEENEIQNFLAELKADVTNQLNLNNGEDEQAFDLLNSFDINNNFDDFVGNFDDTNDNIAQNKSDIDNNKEFEHENDINHDYNDCGTYMDDIYNNNNGDDISRKGSRLKLSDLNDEKNLFPDVNSSFNTPIKSSELKRDSECQTNSPLIFSRSNRTPRKKSVEVILVKKKLKKRKEKESNISFENTTHDDY  Sequence Length: 276 aa Amino acid sequence of Gene PBANKA_050600 (SEQ ID NO: 67)MTDNEDQNKEDLIYYINRYSVNDILGNLEENDKLTNYDENSGICEYEIPFLLENVDNNNNNNTKEHSDRNSVSSYFDDGTCSIISKNDEKHYIDKCEKDKMPKEKINIIFIQNKGEMNSFEDILSMNNASSENLENKLNDRFYQLCCKSIADVNTHNLNKTKNIVKDKKGTLNIEHIDYGDIFLTIRHRLRGREEKTNNMLNNNNNNDNNNNHLYSDMADSVISNWREIKNHENFIKYENYKEHEKEFIRRKLKKKCVNSLNGDKYFMANRKVFDYYRNNLDSYMTNGNEKDICKQENMSLHFLPKKRKSMNNSSLYNSQIIGQNEYILKNRTFLKKFYIKKNFKQQEHIHNDDYYCDDNHSENLYNDDIYNYNKNLSNRQGNLPSNDFIYSCEIQNKKNSIPHNICVDRNVITPRNSTWNNENEIHEEDMVYYHSQNKGKNSHYVEAENEIQSNHYCEDKNTNSFNEYVNEIDKLDENYNMFNKVEEDDNNNNKENFNIYDGDEIDNNEAFDIKIEENDDYETYNNELELEVEVDDGIGNNIPFNNNDNFVNSNKNEDLDNINNCEHVSNSNHTKYGEEDNEQKAPSITSKDDKDYFDLLIKKYEQTRMSINESSTASLSESIYLSKEGTKEPSLNAHEMLKIASNTKNDVNNKIECLNENLIDLKNNKEIINEGECFSNGFSIEKNDIEKENDNIVKLGSVYNNDKTEGERGNIGNKNEKVDLKDSDGYEKLLKNDMYDLYNIKMHDLNNLKSYDFEFSKNLLKNEIFFCGDNIKSDEINLNDNDINEKIDSLMNNYNIMKNKRDKFNEEENEIQNFLAELKADVTNQLNLNNGEDEQAFDLLNSFDINNNFDDFVGNFDDTNDNIAQNKSDIDNNKEFEHENDINHDYNDCGTYMDDIYNNNNGDDISRKGSRLKLSDLNDEKNLFPDVNSSFNTPIKSSELKRDSECQTNSPLIFSRSNRTPRKKSVEVILVKKKLKKRKEKESNISFENTTHDDYTVGTTTATSSINSKRRYPKRNRIKTLRYWIGERELTRRNPETGEIDVVGFSECKNLEELSPHIIGPVYYKKMYLRDVNNLHGKGNEDANNNIDRNDNTDEENEITIEINNGMYENEVYNKIQNKENSVNKNDNVSNILKKSINGSIHNRSDNDAITRNGKKKRKKFINVVNYIKKKTKKKLVKVIDKEVEQENENVDNRNTFSNNDNIINDITNVNHNSQNNLDQNFIAISNDFIENDDNIFFDAISLGDNAHINDIPEKSEEIIEAPGVDAIETTKVNGNEKEINLEKEINLEKEINLEKNKDVHVKKKLLDKKKKKKKKKNKGKEKEIDEMYKQLSFLNFNSFYSKGNEDKSKIEILKKTSTKKKGSKIDKEKVDEENDKHNKNSGKEAKELITKKKKAKNMKKNKKRNMQNKEMKNYYEYTNNEIEKFYNNPNDRIENEYNMGVDLEASIKTEEEKTEKIGELPILNSYTNEQYEHITNTNDITNSKSENFELHKNEDEEVEKLQTSTRRKKKKKSESLIHDTNELNKKRRKTDGNNSGELISINENDEIKNVDADKKINDKEGKYIKKVDKDTIMGSNGNNIDELNKDFEDNDQIKNIKKDEKKKETNTDGSNNMRNINLLEEIDANEKNSTLCLVTHNKKNNTNSQSFIIDKLKSYFNIKELINVKKQKTNNVILNTFENKQIINNNPIRISLSYPSSVELSVENRCNQTRNGQFPLIQKNLSNFKVDINLFCVQIFPNKAHSSNSYDKILIGYIYQGKKVKIYFKNQERYFEKDEFFYIPKYSPFKIVNISRDN CILYVYPINKSequence Length: 1810 aa  Coding Nucleotide sequence of PBANKA_050600(SEQ ID NO: 68)ATGACAGACAACGAGGATCAAAATAAAGAAGATCTGATATATTACATAAATAGATACAGTGTCAATGATATATTGGGAAATTTAGAAGAAAATGATAAGTTAACAAATTATGATGAAAATAGCGGAATATGTGAATATGAAATTCCATTTCTTTTGGAAAATGTCGATAATAATAATAATAATAATACTAAAGAACATTCCGATAGAAATTCTGTATCTAGTTATTTCGATGATGGAACATGTTCGATTATTTCTAAAAATGATGAAAAACATTATATAGACAAATGTGAAAAAGACAAAATGCCAAAGGAAAAAATAAATATTATATTTATTCAGAATAAAGGTGAAATGAATAGCTTTGAAGATATTTTATCCATGAATAATGCAAGCAGTGAAAATTTAGAAAACAAGTTAAATGATAGATTTTATCAACTATGTTGTAAAAGTATTGCTGATGTGAACACCCACAATTTAAATAAAACTAAAAATATTGTAAAAGATAAAAAAGGGACATTGAATATTGAGCATATAGATTATGGTGATATATTTTTAACCATTCGTCATCGTCTAAGAGGGCGTGAAGAAAAAACGAATAACATGCTAAATAATAATAATAATAATGATAATAATAATAATCATTTATATAGTGACATGGCTGATAGTGTTATTAGTAATTGGAGGGAAATAAAAAATCATGAAAATTTTATAAAATATGAAAACTATAAAGAGCATGAAAAGGAGTTTATAAGGAGGAAATTGAAAAAGAAATGCGTCAATAGTTTAAATGGAGATAAATATTTTATGGCCAATAGAAAAGTATTTGATTATTATCGTAATAATTTAGATAGTTACATGACTAATGGGAATGAAAAAGATATATGCAAGCAAGAAAATATGTCTCTACATTTTTTACCAAAAAAGAGAAAATCAATGAATAATAGTTCTTTATACAATTCTCAAATAATTGGACAAAATGAATATATTTTAAAGAATAGAACATTTTTAAAAAAATTTTATATAAAAAAAAATTTTAAGCAACAAGAACATATCCATAATGATGATTATTATTGTGATGATAATCATAGTGAAAATTTATATAATGATGATATATATAATTATAATAAAAACTTGAGTAATAGACAAGGTAATCTACCCAGCAATGATTTTATTTATTCATGTGAAATTCAAAATAAGAAAAATTCAATACCACATAATATATGTGTCGATAGAAATGTAATAACCCCACGGAACAGTACATGGAATAATGAAAACGAAATTCACGAAGAGGATATGGTTTATTATCATTCTCAAAATAAGGGAAAAAATTCACATTATGTAGAAGCAGAAAATGAAATACAATCAAATCATTATTGTGAAGATAAAAATACAAACAGTTTTAACGAATATGTTAATGAAATTGATAAACTCGATGAAAATTATAATATGTTTAACAAAGTTGAAGAGGACGATAATAATAATAACAAAGAAAATTTTAACATTTATGATGGTGATGAAATAGATAATAACGAAGCATTTGATATCAAAATCGAAGAAAATGATGATTATGAAACATATAACAACGAATTAGAATTAGAGGTAGAGGTAGATGATGGAATAGGTAATAATATTCCATTTAATAATAATGATAATTTTGTAAATTCAAATAAGAATGAAGATTTGGATAATATAAATAATTGTGAACATGTTTCAAATTCAAATCATACAAAATATGGGGAAGAAGACAATGAGCAAAAAGCTCCATCAATAACCAGTAAAGATGATAAAGATTATTTTGATTTACTAATAAAAAAATATGAACAAACTAGAATGTCAATTAATGAATCTAGTACAGCCTCACTTAGTGAAAGTATTTATTTATCAAAAGAAGGAACAAAAGAACCTTCTTTAAATGCTCACGAAATGTTAAAAATCGCATCTAACACAAAGAATGATGTAAATAATAAAATTGAATGTTTGAATGAAAACTTAATAGATTTAAAAAATAACAAGGAAATTATTAATGAAGGGGAATGTTTTAGTAATGGTTTTTCTATCGAAAAAAATGACATAGAAAAGGAAAATGATAATATAGTAAAATTAGGAAGTGTATATAATAATGACAAAACAGAGGGGGAAAGAGGGAATATTGGAAACAAAAATGAAAAAGTAGACCTTAAAAGATAGTGATGGATATGAGAAATTATTAAAAAATGACATGTACGATTTATATAATATTAAGATGCATGATTTAAATAACTTAAAATCATATGATTTTGAATTTTCAAAAAATTTATTAAAAAACGAGATTTTTTTTTGTGGTGATAATATAAAAAGTGATGAAATAAATTTAAATGATAATGACATAAATGAAAAGATTGATTCACTAATGAACAATTACAATATTATGAAAAACAAACGTGACAAATTTAATGAAGAAGAAAACGAAATTCAAAACTTTTTAGCAGAATTAAAAGCTGATGTAACTAATCAACTCAATCTAAATAACGGGGAAGATGAACAGGCTTTTGATTTGCTTAATTCGTTTGATATAAACAATAACTTTGACGATTTTGTTGGCAACTTTGATGATACAAATGATAACATAGCTCAAAATAAATCAGACATAGACAATAATAAAGAGTTCGAACACGAAAATGATATAAATCATGATTATAACGATTGTGGTACATATATGGATGATATATATAATAACAATAATGGTGATGATATTTCGAGAAAGGGATCACGTCTGAAATTGTCTGATTTAAATGACGAAAAGAATTTATTTCCAGATGTCAACTCCTCTTTTAATACTCCTATAAAATCTTCTGAACTAAAGAGAGATTCAGAATGCCAAACAAATTCACCACTTATATTTTCTAGAAGTAATAGAACTCCTAGGAAAAAAAGTGTAGAAGTAATATTAGTAAAGAAAAAATTAAAAAAAAGAAAAGAAAAAGAATCAAATATATCATTTGAAAATACAACACATGATGATTATACTGTTGGTACAACTACTGCTACTAGTAGCATCAATTCGAAAAGAAGATATCCTAAAAGAAATAGAATAAAAACGTTGCGATACTGGATAGGTGAAAGGGAACTTACTAGAAGAAATCCTGAAACAGGCGAAATAGATGTTGTAGGTTTTAGTGAATGCAAAAATTTAGAAGAATTATCTCCTCATATTATTGGTCCAGTTTATTATAAAAAAATGTATTTACGAGATGTGAATAATTTACATGGAAAAGGAAACGAAGATGCTAACAACAATATAGATAGAAATGATAATACTGATGAAGAAAATGAAATAACGATAGAAATCAATAATGGAATGTATGAAAATGAAGTGTATAATAAAATTCAGAATAAAGAGAATTCTGTGAATAAAAATGATAATGTTAGTAACATATTGAAAAAAAGTATAAATGGTAGCATTCATAATAGAAGTGATAATGATGCAATAACTAGAAATGGGAAAAAGAAAAGAAAAAAGTTTATTAATGTTGTTAATTATATTAAAAAAAAAACAAAAAAAAAATTAGTCAAAGTTATAGATAAAGAAGTAGAGCAGGAAAATGAAAATGTAGATAATCGTAACACTTTTTCAAATAATGATAATATAATTAATGACATAACAAATGTCAATCACAATTCTCAAAATAATTTGGATCAAAATTTTATTGCAATTAGTAATGATTTTATTGAAAATGATGACAATATTTTTTTCGATGCGATTAGTCTTGGCGATAATGCTCACATAAATGATATTCCAGAAAAAAGCGAAGAAATTATTGAAGCACCAGGAGTAGATGCAATTGAAACGACTAAAGTTAATGGAAACGAAAAGGAAATCAATTTAGAAAAGGAAATCAATTTAGAAAAGGAAATCAATTTAGAAAAGAATAAAGATGTACATGTGAAAAAGAAATTATTAGATAAAAAGAAAAAGAAAAAAAAAAAGAAAAACAAGGGAAAAGAAAAGGAAATAGACGAAATGTACAAGCAATTATCATTTTTGAATTTTAATTCGTTTTATTCTAAAGGAAATGAAGATAAATCAAAAATAGAAATTTTGAAAAAAACAAGTACCAAAAAAAAAGGGAGTAAAATTGATAAAGAAAAGGTAGATGAGGAAAATGATAAACATAATAAAAATTCGGGAAAGGAAGCCAAAGAATTAATTACAAAAAAAAAGAAAGCCAAGAATATGAAGAAAAATAAAAAGAGAAATATGCAGAATAAAGAAATGAAAAATTATTATGAATATACAAATAATGAAATCGAAAAGTTCTACAACAATCCAAATGATAGAATAGAGAATGAATACAATATGGGAGTCGATTTAGAAGCATCAATAAAAACTGAAGAAGAAAAAACAGAAAAAATTGGAGAGTTGCCCATTTTAAATTCATATACTAATGAGCAATATGAGCACATAACGAATACAAATGATATAACAAATTCGAAAAGTGAAAATTTTGAACTCCACAAAAATGAAGACGAAGAAGTGGAAAAGCTACAAACTTCTACACGTCGAAAAAAGAAAAAAAAAAGTGAAAGTTTAATTCATGATACAAATGAATTGAATAAAAAGCGAAGAAAAACAGATGGAAATAATTCAGGGGAATTAATTTCTATTAATGAAAATGATGAGATAAAAAATGTAGATGCTGATAAAAAAATAAATGACAAAGAAGGTAAATATATAAAGAAAGTTGACAAGGATACAATTATGGGATCAAATGGAAATAATATTGATGAATTAAATAAGGATTTTGAAGATAATGATCAAATTAAAAATATAAAAAAAGATGAAAAAAAAAAAGAGACAAATACAGATGGTTCTAATAATATGAGAAATATAAATTTATTAGAAGAAATAGATGCAAATGAAAAAAATAGTACATTATGTTTGGTAACTCACAATAAAAAAAATAATACGAATAGTCAAAGTTTTATTATAGATAAATTAAAATCGTATTTCAATATAAAAGAGTTAATAAATGTCAAAAAACAAAAAACAAATAATGTAATATTAAATACTTTTGAAAATAAACAAATAATAAATAATAATCCTATACGTATTTCTCTTTCCTATCCTTCTAGTGTAGAATTATCAGTTGAAAATAGATGCAACCAAACAAGAAATGGACAATTTCCACTTATACAAAAGAACTTAAGCAACTTCAAGGTAGACATAAATTTATTTTGTGTTCAAATTTTCCCAAACAAAGCACATAGCTCGAATAGTTATGATAAAATTTTGATTGGGTATATATATCAGGGAAAAAAGGTAAAGATTTATTTTAAGAACCAAGAAAGATATTTTGAAAAGGATGAGTTTTTTTACATACCCAAATACTCTCCTTTCAAAATTGTCAACATAAGCAGGGACAATTGTATTTTATATGTTTATCCAATAAATAAATAA  Sequence Length: 5434 bp SERA5 (serine repeat antigen 5) PlasmoDB ID: PF3D7_0207600Chromosome 2; position 303,593-307,027Full Sequence: base pairs 1-2994 (excluding introns) (SEQ ID NO: 69)ATGAAGTCATATATTTCCTTGTTTTTCATATTGTGTGTTATATTTAACAAAAATGTTATAAAATGTACAGGAGAAAGTCAAACAGGTAATACAGGAGGAGGTCAAGCAGGTAATACAGGAGGAGATCAAGCAGGTAGTACAGGAGGAAGTCCACAAGGTAGTACGGGAGCAAGTCCACAAGGTAGTACGGGAGCAAGTCCACAAGGTAGTACGGGAGCAAGTCAACCCGGAAGTTCCGAACCAAGCAATCCTGTAAGTTCCGGACATTCTGTAAGTACTGTATCAGTATCACAAACTTCAACTTCTTCAGAAAAACAGGATACAATTCAAGTAAAATCAGCTTTATTAAAAGATTATATGGGTTTAAAAGTTACTGGTCCATGTAACGAAAATTTCATAATGTTCTTAGTTCCTCATATATATATTGATGTTGATACAGAAGATACTAATATCGAATTAAGAACAACATTGAAAAAAACAAATAATGCAATATCATTTGAATCAAACAGTGGTTCATTAGAAAAAAAAAAATATGTAAAACTACCATCAAATGGTACAACTGGTGAACAAGGTTCAAGTACGGGAACAGTTAGAGGAGATACAGAACCAATTTCAGATTCAAGCTCAAGTTCAAGTTCAAGCTCTAGTTCAAGTTCAAGTTCAAGTTCAAGTTCTAGTTCAAGTTCTAGTTCAAGTTCAGAAAGTCTTCCTGCTAATGGACCTGATTCCCCTACTGTTAAACCGCCAAGAAATTTACAAAATATATGTGAAACTGGAAAAAACTTCAAGTTGGTAGTATATATTAAGGAGAATACATTAATACTTAAATGGAAAGTATACGGAGAAACAAAAGATACTACTGAAAATAACAAAGTTGATGTAAGAAAGTATTTGATAAATGAAAAGGAAACCCCATTTACTAATATACTAATACATGCGTATAAAGAACATAATGGAACAAACTTAATAGAAAGTAAAAACTACGCAATAGGATCAGACATTCCAGAAAAATGTGATACCTTAGCTTCCAATTGCTTTTTAAGTGGTAATTTTAACATTGAAAAATGCTTTCAATGTGCTCTTTTAGTAGAAAAAGAAAATAAAAATGACGTATGTTACAAATACCTATCTGAAGATATTGTAAGTAAATTCAAAGAAATAAAAGCTGAGACAGAAGATGATGATGAAGATGATTATACTGAATATAAATTAACAGAATCTATTGATAATATATTAGTAAAAATGTTTAAAACAAATGAAAATAATGATAAATCAGAATTAATAAAATTAGAAGAAGTAGATGATAGTTTGAAATTAGAATTAATGAATTACTGTAGTTTACTTAAAGACGTAGATACAACAGGTACCTTAGATAATTATGGGATGGGAAATGAAATGGATATATTTAATAACTTAAAGAGATTATTAATTTATCATTCAGAAGAAAATATTAATACTTTAAAAAATAAATTCCGTAATGCAGCTGTATGTCTTAAAAATGTTGATGATTGGATTGTAAATAAGAGAGGTTTAGTATTACCTGAATTAAATTATGATTTAGAATATTTCAATGAACATTTATATAATGATAAAAATTCTCCAGAAGATAAAGATAATAAAGGAAAAGGTGTCGTACATGTTGATACAACTTTAGAAAAAGAAGATACTTTATCATATGATAACTCAGATAATATGTTTTGTAATAAAGAATATTGTAACAGATTAAAAGATGAAAATAATTGTATATCTAATCTTCAAGTTGAAGATCAAGGTAATTGTGATACTTCATGGATTTTTGCTTCAAAATATCATTTAGAAACTATTAGATGTATGAAAGGATATGAACCTACCAAAATTTCTGCTCTTTATGTAGCTAATTGTTATAAAGGTGAACATAAAGATAGATGTGATGAAGGTTCTAGTCCAATGGAATTCTTACAAATTATTGAAGATTATGGATTCTTACCAGCAGAATCAAATTATCCATATAACTATGTGAAAGTTGGAGAACAATGTCCAAAGGTAGAAGATCACTGGATGAATCTATGGGATAATGGAAAAATCTTACATAACAAAAATGAACCTAATAGTTTAGATGGTAAGGGATATACTGCATATGAAAGTGAAAGATTTCATGATAATATGGATGCATTTGTTAAAATTATTAAAACTGAAGTAATGAATAAAGGTTCAGTTATTGCATATATTAAAGCTGAAAATGTTATGGGATATGAATTTAGTGGAAAGAAAGTACAGAACTTATGTGGTGATGATACAGCTGATCATGCAGTTAATATTGTTGGTTATGGTAATTATGTGAATAGCGAAGGAGAAAAAAAATCCTATTGGATTGTAAGAAACAGTTGGGGTCCATATTGGGGAGATGAAGGTTATTTTAAAGTAGATATGTATGGACCAACTCATTGTCATTTTAACTTTATTCACAGTGTTGTTATATTCAATGTTGATTTACCTATGAATAATAAAACAACTAAAAAAGAATCAAAAATATATGATTATTATTTAAAGGCCTCTCCAGAATTTTATCATAACCTTTACTTTAAGAATTTTAATGTTGGTAAGAAAAATTTATTCTCTGAAAAGGAAGATAATGAAAACAACAAAAAATTAGGTAACAACTATATTATATTCGGTCAAGATACGGCAGGATCAGGACAAAGTGGAAAGGAAAGCAATACTGCATTAGAATCTGCAGGAACTTCAAATGAAGTCTCAGAACGTGTTCATGTTTATCACATATTAAAACATATAAAGGATGGCAAAATAAGAATGGGTATGCGTAAATATATAGATACACAAGATGTAAATAAGAAACATTCTTGTACAAGATCCTATGCATTTAATCCAGAGAATTATGAAAAATGTGTAAATTTATGTAATGTGAACTGGAAAACATGCGAGGAAAAAACATCACCAGGACTTTGTTTATCCAAATTGGATACAAATAACGAATGTTATTTCTGTTATGTATAA Full Sequence: 1-997 amino acids (SEQ ID NO: 70)MKSYISLFFILCVIFNKNVIKCTGESQTGNTGGGQAGNTGGDQAGSTGGSPQGSTGASPQGSTGASPQGSTGASQPGSSEPSNPVSSGHSVSTVSVSQTSTSSEKQDTIQVKSALLKDYMGLKVTGPCNENFIMFLVPHIYIDVDTEDTNIELRTTLKKTNNAISFESNSGSLEKKKYVKLPSNGTTGEQGSSTGTVRGDTEPISDSSSSSSSSSSSSSSSSSSSSSSSSSSSESLPANGPDSPTVKPPRNLQNICETGKNFKLVVYIKENTLILKWKVYGETKDTTENNKVDVRKYLINEKETPFTNILIHAYKEHNGTNLIESKNYAIGSDIPEKCDTLASNCFLSGNFNIEKCFQCALLVEKENKNDVCYKYLSEDIVSKFKEIKAETEDDDEDDYTEYKLTESIDNILVKMFKTNENNDKSELIKLEEVDDSLKLELMNYCSLLKDVDTTGTLDNYGMGNEMDIFNNLKRLLIYHSEENINTLKNKFRNAAVCLKNVDDWIVNKRGLVLPELNYDLEYFNEHLYNDKNSPEDKDNKGKGVVHVDTTLEKEDTLSYDNSDNMFCNKEYCNRLKDENNCISNLQVEDQGNCDTSWIFASKYHLETIRCMKGYEPTKISALYVANCYKGEHKDRCDEGSSPMEFLQIIEDYGELPAESNYPYNYVKVGEQCPKVEDHWMNLWDNGKILHNKNEPNSLDGKGYTAYESERFHDNMDAFVKIIKTEVMNKGSVIAYIKAENVMGYEFSGKKVQNLCGDDTADHAVNIVGYGNYVNSEGEKKSYWIVRNSWGPYWGDEGYEKVDMYGPTHCHFNFIHSVVIFNVDLPMNNKTTKKESKIYDYYLKASPEFYHNLYFKNFNVGKKNLFSEKEDNENNKKLGNNYIIFGQDTAGSGQSGKESNTALESAGTSNEVSERVHVYHILKHIKDGKIRMGMRKYIDTQDVNKKHSCTRSYAFNPENYEKCVNLCNVNWKTCEEKTSPGLCLSKLDTNNECYFCYV Y2H Clone name: 1 7-1 (nucleotides 2433-2994; amino acids 561 base pairs(SEQ ID NO: 71)AACTTTATTCACAGTGTTGTTATATTCAATGTTGATTTACCTATGAATAATAAAACAACTAAAAAAGAATCAAAAATATATGATTATTATTTAAAGGCCTCTCCAGAATTTTATCATAACCTTTACTTTAAGAATTTTAATGTTGGTAAGAAAAATTTATTCTCTGAAAAGGAAGATAATGAAAACAACAAAAAATTAGGTAACAACTATATTATATTCGGTCAAGATACGGCAGGATCAGGACAAAGTGGAAAGGAAAGCAATACTGCATTAGAATCTGCAGGAACTTCAAATGAAGTCTCAGAACGTGTTCATGTTTATCACATATTAAAACATATAAAGGATGGCAAAATAAGAATGGGTATGCGTAAATATATAGATACACAAGATGTAAATAAGAAACATTCTTGTACAAGATCCTATGCATTTAATCCAGAGAATTATGAAAAATGTGTAAATTTATGTAATGTGAACTGGAAAACATGCGAGGAAAAAACATCACCAGGACTTTGTTTATCCAAATTGGATACAAATAACGAATGTTATTTCTGTTATGTATAA  186 amino acids(SEQ ID NO: 72)NFIHSVVIFNVDLPMNNKTTKKESKIYDYYLKASPEFYHNLYFKNFNVGKKNLFSEKEDNENNKKLGNNYIIFGQDTAGSGQSGKESNTALESAGTSNEVSERVHVYHILKHIKDGKIRMGMRKYIDTQDVNKKHSCTRSYAFNPENYEKCVNLCNVNWKTCEEKTSPGLCLSKLDTNNECYFCYVSUB1 (subtilisin-like protease 1) PlasmoDB ID: PF3D7_0507500Chromosome 5; position 307,490-309,556Full Sequence: base pairs 1-2067 (excluding introns) (SEQ ID NO: 73)ATGATGCTCAATAAAAAAGTTGTTGCTTTGTGCACACTTACCTTACATCTTTTTTGTATATTTCTATGTCTAGGAAAGGAAGTAAGGTCTGAAGAAAATGGGAAAATACAAGATGATGCTAAAAAGATTGTTAGCGAATTACGATTCCTAGAAAAAGTAGAAGATGTTATTGAAAAGAGTAACATAGGAGGGAATGAGGTAGATGCCGATGAAAATTCATTTAATCCGGATACTGAGGTTCCCATAGAAGAGATAGAAGAAATAAAAATGAGGGAACTGAAAGATGTAAAGGAAGAAAAAAATAAAAATGACAACCATAATAATAATAATAATAATATTAGTAGTAGTAGTAGTAGTAGTAGTAATACTTTTGGTGAAGAAAAAGAAGAAGTATCTAAGAAAAAAAAAAAGTTAAGACTTATAGTTAGCGAGAATCATGCAACTACCCCCTCGTTTTTCCAAGAATCCCTTTTAGAACCTGATGTTTTATCCTTTTTAGAAAGTAAAGGGAATTTGTCCAACTTGAAAAATATCAATTCTATGATTATAGAACTAAAGGAAGATACAACGGATGATGAATTAATATCTTATATTAAAATTCTTGAGGAGAAGGGAGCTTTGATTGAATCAGATAAATTAGTGAGTGCAGATAATATTGATATAAGTGGTATAAAAGATGCTATAAGAAGAGGTGAAGAAAATATTGATGTTAATGATTATAAAAGTATGTTAGAAGTCGAAAATGATGCTGAAGATTATGATAAAATGTTTGGTATGTTTAATGAATCACATGCTGCAACATCTAAAAGGAAACGCCATTCAACAAATGAGCGTGGATATGATACATTTTCATCACCTTCATATAAGACATATTCAAAAAGTGATTATTTATATGATGATGATAATAATAATAATAATTATTATTATAGTCATAGTAGTAATGGTCATAATAGTAGTAGTCGTAATAGTAGTAGTAGTCGTAGTAGACCAGGTAAATATCATTTCAATGATGAATTTCGTAATTTGCAATGGGGTTTAGATTTATCCAGATTAGATGAAACACAAGAATTAATTAACGAACATCAAGTGATGAGTACTCGTATATGTGTTATAGATAGTGGTATTGATTATAATCATCCCGATTTAAAAGATAATATTGAATTAAATTTAAAAGAATTACATGGAAGGAAAGGTTTTGATGATGATAATAATGGTATAGTTGATGATATATATGGTGCTAATTTTGTAAATAATTCAGGAAACCCGATGGATGATAATTATCATGGTACTCATGTATCAGGAATTATATCTGCCATAGGAAATAATAATATAGGTGTTGTAGGTGTTGATGTAAATTCAAAATTAATTATTTGTAAAGCATTAGATGAACATAAATTAGGAAGATTAGGAGATATGTTCAAATGTTTAGATTATTGTATAAGTAGAAATGCACATATGATAAATGGAAGCTTTTCATTTGATGAATATAGTGGTATTTTTAATTCTTCTGTAGAATATTTACAAAGAAAAGGTATCCTCTTTTTTGTATCTGCAAGTAATTGTAGTCATCCTAAATCGTCAACACCAGATATTAGAAAATGTGATTTATCCATAAATGCAAAATATCCCCCTATCTTATCTACTGTTTATGATAATGTTATATCTGTTGCTAATTTAAAAAAAAATGATAATAATAATCATTATTCATTATCCATTAATTCTTTTTATAGCAATAAATATTGTCAACTAGCTGCACCAGGAACTAATATATATTCTACTGCTCCACATAATTCATATCGAAAATTAAATGGTACATCTATGGCTGCTCCACATGTAGCTGCAATAGCATCACTCATATTTTCTATTAATCCTGACTTATCATATAAAAAAGTTATACAAATATTAAAAGATTCTATTGTATATCTCCCTTCCTTAAAAAATATGGTTGCATGGGCAGGATATGCAGATATAAATAAGGCAGTCAATTTAGCCATAAAATCAAAAAAAACATATATCAATTCTAATATATCTAACAAGTGGAAAAAAAAAAGTAGATATTTGCATTAA  Full Sequence: 1-688 amino acids (SEQ ID NO: 74)MMLNKKVVALCTLTLHLFCIFLCLGKEVRSEENGKIQDDAKKIVSELRFLEKVEDVIEKSNIGGNEVDADENSFNPDTEVPIEEIEEIKMRELKDVKEEKNKNDNHNNNNNNISSSSSSSSNTFGEEKEEVSKKKKKLRLIVSENHATTPSFFQESLLEPDVLSFLESKGNLSNLKNINSMIIELKEDTTDDELISYIKILEEKGALIESDKLVSADNIDISGIKDAIRRGEENIDVNDYKSMLEVENDAEDYDKMFGMFNESHAATSKRKRHSTNERGYDTFSSPSYKTYSKSDYLYDDDNNNNNYYYSHSSNGHNSSSRNSSSSRSRPGKYHFNDEFRNLQWGLDLSRLDETQELINEHQVMSTRICVIDSGIDYNHPDLKDNIELNLKELHGRKGFDDDNNGIVDDIYGANFVNNSGNPMDDNYHGTHVSGIISAIGNNNIGVVGVDVNSKLIICKALDEHKLGRLGDMFKCLDYCISRNAHMINGSFSFDEYSGIFNSSVEYLQRKGILFFVSASNCSHPKSSTPDIRKCDLSINAKYPPILSTVYDNVISVANLKKNDNNNHYSLSINSFYSNKYCQLAAPGTNIYSTAPHNSYRKLNGTSMAAPHVAAIASLIFSINPDLSYKKVIQILKDSIVYLPSLKNMVAWAGYADINKAVNLAIKSKKTYINSNISNKWKKKSRYLH PKG (cGMP-dependent protein kinase) PlasmoDB ID: PF3D7_1436600Chromosome 14; position 1,490,654-1,494,214Full Sequence: base pairs 1-2562 (excluding introns) (SEQ ID NO: 75)ATGGAAGAAGATGATAATCTAAAAAAAGGGAATGAAAGAAATAAAAAGAAGGCTATATTTTCAAATGATGATTTTACAGGAGAAGATAGTTTAATGGAGGATCATTTAGAACTTCGGGAAAAGCTTTCAGAAGATATTGATATGATAAAGACTTCCTTAAAAAATAATCTAGTTTGTAGTACATTAAACGATAATGAAATATTGACTCTGTCTAATTATATGCAATTCTTTGTTTTTAAAAGTGGAAATTTAGTAATAAAACAAGGGGAAAAAGGGTCATACTTTTTCATTATTAATAGTGGCAAATTTGACGTTTATGTAAATGATAAAAAAGTAAAGACTATGGGAAAAGGTAGTTCTTTCGGTGAAGCTGCTTTAATTCATAATACCCAAAGAAGTGCAACTATTATTGCAGAAACTGATGGAACTCTATGGGGAGTTCAAAGAAGTACATTTAGAGCTACCCTAAAACAATTATCTAATAGAAATTTTAACGAAAACAGAACATTTATCGATTCCGTTTCAGTTTTTGATATGTTAACTGAAGCACAAAAAAACATGATTACTAATGCTTGTGTAATACAAAACTTTAAATCTGGTGAAACCATTGTTAAACAAGGAGATTATGGAGATGTCTTATACATTTTGAAAGAAGGAAAGGCTACAGTATATATTAACGATGAAGAGATAAGGGTTTTAGAGAAAGGTTCCTATTTTGGGGAAAGAGCTCTACTGTATGATGAACCAAGAAGTGCAACAATCATTGCAAAAGAACCAACCGCTTGTGCATCCATTTGTAGGAAATTATTAAATATTGTTCTAGGAAACTTACAAGTAGTTTTATTTCGTAATATTATGACTGAAGCTTTACAACAGAGTGAAATTTTTAAACAATTTAGTGGGGATCAATTAAACGATTTAGCAGATACCGCCATTGTTCGAGATTATCCAGCTAATTATAATATATTACATAAGGATAAGGTAAAATCCGTTAAATATATTATTGTATTGGAAGGTAAAGTAGAATTATTTCTTGATGATACTTCTATTGGTATATTATCCAGAGGAATGTCTTTTGGAGATCAATATGTATTAAATCAGAAACAACCATTTAAGCATACTATTAAATCATTAGAAGTTTGTAAAATCGCATTAATAACGGAAACTTGTTTAGCTGATTGTCTAGGAAATAATAATATTGATGCATCTATTGATTATAATAATAAAAAAAGTATTATAAAGAAAATGTATATCTTTAGATACTTAACTGATAAACAATGTAATTTATTAATTGAAGCTTTTAGAACCACAAGATATGAAGAAGGTGATTATATAATACAAGAAGGAGAAGTAGGATCTAGATTTTATATAATAAAAAATGGAGAAGTAGAAATAGTAAAAAATAAAAAAAGGTTACGTACCTTAGGAAAGAATGATTACTTTGGTGAAAGAGCTTTATTATATGATGAACCAAGAACAGCTTCTGTTATAAGTAAAGTAAATAATGTTGAATGTTGGTTTGTTGATAAAAGTGTGTTTTTACAAATTATACAAGGACCTATGTTAGCACATTTGGAAGAAAGAATAAAAATGCAAGATACTAAAGTAGAAATGGATGAACTAGAAACAGAACGAATTATTGGAAGAGGTACTTTCGGAACAGTTAAATTAGTTCATCATAAACCAACAAAAATAAGATATGCTTTAAAATGTGTTAGTAAAAGAAGTATTATTAATTTAAATCAACAAAACAATATAAAATTAGAAAGAGAAATAACAGCAGAAAATGATCATCCATTTATTATAAGATTAGTAAGAACATTTAAAGATTCTAAATATTTCTATTTTCTAACAGAATTAGTAACAGGTGGAGAATTATATGATGCTATTAGAAAATTAGGTTTATTATCTAAATCACAAGCTCAATTTTATTTAGGTTCTATCATTTTAGCTATTGAATATTTACATGAAAGAAATATTGTATATAGAGATTTAAAACCAGAAAACATTTTATTAGATAAACAAGGTTATGTAAAACTAATCGATTTTGGTTGTGCCAAAAAGGTACAAGGTAGAGCTTATACATTAGTAGGTACACCTCATTATATGGCACCTGAGGTTATTTTAGGAAAAGGTTATGGATGTACTGTTGACATATGGGCATTGGGAATATGCCTATATGAATTTATATGTGGTCCATTACCATTTGGTAATGATGAAGAAGATCAATTAGAAATTTTCCGTGATATATTAACCGGCCAACTTACATTTCCAGATTATGTAACAGACACAGATAGCATAAATTTGATGAAAAGACTTCTATGTAGATTACCTCAAGGAAGAATTGGTTGTTCAATAAATGGCTTCAAAGACATAAAGGATCACCCATTTTTCTCAAACTTTAATTGGGATAAATTGGCTGGTCGTTTGCTTGATCCGCCTTTAGTATCAAAAAGTGAAACTTATGCAGAAGATATTGATATTAAACAAATAGAGGAGGAGGATGCTGAGGATGATGAGGAACCATTGAACGATGAAGACAACTGGGACATAGATTTTTAA Full Sequence: 1-853 amino acids (SEQ ID NO: 76)MEEDDNLKKGNERNKKKAIFSNDDFTGEDSLMEDHLELREKLSEDIDMIKTSLKNNLVCSTLNDNEILTLSNYMQFFVFKSGNLVIKQGEKGSYFFIINSGKFDVYVNDKKVKTMGKGSSFGEAALIHNTQRSATIIAETDGTLWGVQRSTFRATLKQLSNRNFNENRTFIDSVSVFDMLTEAQKNMITNACVIQNFKSGETIVKQGDYGDVLYILKEGKATVYINDEEIRVLEKGSYFGERALLYDEPRSATITAKEPTACASICRKLLNIVLGNLQVVLFRNIMTEALQQSEIFKQFSGDQLNDLADTAIVRDYPANYNILHKDKVKSVKYIIVLEGKVELFLDDTSIGILSRGMSFGDQYVLNQKQPFKHTIKSLEVCKIALITETCLADCLGNNNIDASIDYNNKKSIIKKMYIFRYLTDKQCNLLIEAFRTTRYEEGDYIIQEGEVGSRFYIIKNGEVEIVKNKKRLRTLGKNDYFGERALLYDEPRTASVISKVNNVECWFVDKSVFLQIIQGPMLAHLEERIKMQDTKVEMDELETERIIGRGTFGTVKLVHHKPTKIRYALKCVSKRSIINLNQQNNIKLEREITAENDHPFIIRLVRTFKDSKYFYFLTELVTGGELYDAIRKLGLLSKSQAQFYLGSIILAIEYLHERNIVYRDLKPENILLDKQGYVKLIDFGCAKKVQGRAYTLVGTPHYMAPEVILGKGYGCTVDIWALGICLYEFICGPLPFGNDEEDQLEIFRDILTGQLTFPDYVTDTDSINLMKRLLCRLPQGRIGCSINGFKDIKDHPFFSNFNWDKLAGRLLDPPLVSKSETYAEDIDIKQIEEEDAEDDEEPLNDEDNWDIDF 

Underlined amino acid sequences and cDNA nucleic acid sequencescorrespond to immunorelevant regions of the gene products and nucleicacids encoding them. The antigenic fragments (polypeptides) wereidentified by virtue of binding of antibodies from patients that areresistant to malaria.

The invention encompasses “fragments” and “peptides” of SEQ ID NOs: 2,3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39,42, or 43, 47, 67, 70, 74, or 76 preferably, the clone 2 polypeptide orthe PF10_0212a polypeptide (a.k.a., PfSEP-1A; SEQ ID NO:2) describedherein. Such peptides represent portions of the polypeptide that have,for example, specific immunogenic or binding properties. A fragment canbe between 3-10 amino acids, 10-20 amino acids, 20-40 amino acids, 40-56amino acids in length or even longer. Amino acid sequences having atleast 70% amino acid identity, preferably at least 80% amino acididentity, more preferably at least 90% identity, and most preferably 95%identity to the fragments described herein are also included within thescope of the present invention.

Furthermore, the present invention encompasses fragments and derivativesof the nucleic acid sequences of the present invention, as well asfragments and portions of the amino acid sequences of the presentinvention.

A “polynucleotide” is a nucleic acid polymer of ribonucleic acid (RNA),deoxyribonucleic acid (DNA), modified RNA or DNA, or RNA or DNA mimetics(such as PNAs), and derivatives thereof, and homologues thereof. Thus,polynucleotides include polymers composed of naturally occurringnucleobases, sugars and covalent inter-nucleoside (backbone) linkages aswell as polymers having non-naturally-occurring portions that functionsimilarly. Such modified or substituted nucleic acid polymers are wellknown in the art and for the purposes of the present invention, arereferred to as “analogues.” Oligonucleotides are generally shortpolynucleotides from about 10 to up to about 160 or 200 nucleotides.

A “variant polynucleotide” or a “variant nucleic acid sequence” means apolynucleotide having at least about 60% nucleic acid sequence identity,more preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% nucleic acid sequence identity and yet more preferably at leastabout 99% nucleic acid sequence identity with the nucleic acid sequenceof SEQ ID NOs: 1, 4, 5, 8, 9, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29,32, 33, 36, 37, 40, 41, 44, 45, or 48, preferably SEQ ID NO: 1. Variantsdo not encompass the native nucleotide sequence. Other variantpolynucleotides include those that differ from SEQ ID NO: 1, but becauseof the redundancy of the genetic code, encode a polypeptide of SEQ IDNo: 2, or amino acids 2-50 of SEQ ID No: 2, fragments of variantsthereof.

Ordinarily, variant polynucleotides are at least about 8 nucleotides inlength, often at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29 30, 35, 40, 45, 50, 55, 60nucleotides in length, or even about 75-200 nucleotides in length, ormore.

In general, a polypeptide variant preserves antigenic function andincludes any variant in which residues at a particular position in thesequence have been substituted by other amino acids, and furtherincludes the possibility of inserting an additional residue or residuesbetween two residues of the parent polypeptide as well as thepossibility of deleting one or more residues from the parent sequence.comprising

“A polypeptide variant” means a polypeptide having at least about 70%amino acid sequence identity with an amino acid sequence of SEQ ID NOs:2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38,39, 42, 43, 46, or 47, preferably SEQ ID NO:2 or SEQ ID NO:3. Forexample, polypeptide variants include those wherein one or more aminoacid residues are added or deleted at the N- or C-terminus of thefull-length native amino acid sequence. A polypeptide variant will haveat least about 71%-75% amino acid sequence identity; at least about76%-79% amino acid sequence identity; at least about 80% amino acidsequence identity, at least about 81% amino acid sequence identity, atleast about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% amino acid sequence identity and at least about99% amino acid sequence identity with a full-length sequence.Ordinarily, variant polypeptides are at least about 10 amino acids inlength, often at least about 20 amino acids in length, more often atleast about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 aminoacids in length, or more.

Useful conservative substitutions are shown in Table 2 below.Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution does not materiallyalter the biological activity of the compound.

TABLE 2 Exemplary substitutions Origional Preferred residue Exemplarysubstitutions substitutions Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) AspAsp Gly (G) Pro, Ala Ala His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu,Val, Met, Ala, Phe, Norleucine Leu Leu (L) Norleucine, Ile, Val, Met,Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F)Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) SerSer Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile,Leu, Met, Phe, Ala, Norleucine Leu

The polypeptides of the invention can be either synthesized in vitro orexpressed recombinantly from the polynucleotide sequences. Because ofredundancy in the genetic code, the sequences need not be identical topractice the invention. Polynucleotide and polypeptide sequenceidentities can be from 70%-100%, such as 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% and of course, 100%.

The polypeptides of the invention can be readily synthesized in vitrousing polypeptide chemistry. For example, polypeptide synthesis can becarried out in a stepwise manner on a solid phase support using anautomated polypeptide synthesizer, such as a Rainin Symphony PeptideSynthesizer, Advanced Chemtech Peptide Synthesizer, Argonaut ParallelSynthesis System, or an Applied Biosystems Peptide Synthesizer. Thepeptide synthesizer instrument combines the Fmoc chemistry withHOBt/HBTU/DIEA activation to perform solid-phase peptide synthesis.

The side chains of many amino acids contain chemically reactive groups,such as amines, alcohols, or thiols. These side chains must beadditionally protected to prevent undesired side-reactions during thecoupling step. Side chain protecting groups that are base-stable, morepreferably, both base-stabile and acid-labile are most useful.

“Percent (%) nucleic acid sequence identity” with respect to nucleicacid sequences is defined as the percentage of nucleotides in acandidate sequence that are identical with the nucleotides in thesequence of interest, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining % nucleic acid sequence identitycan be achieved in various ways that are within the skill in the art,for instance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

“Consisting essentially of a polynucleotide having a % sequenceidentity” means that the polynucleotide does not substantially differ inlength, but may differ substantially in sequence. Thus, a polynucleotide“A” consisting essentially of a polynucleotide having at least 80%sequence identity to a known sequence “B” of 100 nucleotides means thatpolynucleotide “A” is about 100 nts long, but up to 20 nts can vary fromthe “B” sequence. The polynucleotide sequence in question can be longeror shorter due to modification of the termini, such as, for example, theaddition of 1-15 nucleotides to produce specific types of probes,primers and other molecular tools, etc., such as the case of whensubstantially non-identical sequences are added to create intendedsecondary structures. Such non-identical nucleotides are not consideredin the calculation of sequence identity when the sequence is modified by“consisting essentially of.”

Vaccine Compositions

The present invention is further directed to an immunogenic composition,e.g., a vaccine composition capable of blocking P. falciparum infection,for example a peptide vaccine or a DNA vaccine capable of blockingSchizont rupture at blood stage infection. The vaccine compositioncomprises one or more of the polypeptides, the nucleic acid sequences,or antigens thereof, as described herein.

A person skilled in the art will be able to select preferred peptides,polypeptides, nucleic acid sequences or combination of thereof bytesting, e.g., the blocking of the Schizont rupture or parasite egressfrom RBCs in vitro. Peptide(s) with the desired activity are thencombined as a vaccine. A suitable vaccine will preferably containbetween 1 and 20 peptides, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different peptides, furtherpreferred 6, 7, 8, 9, 10 11, 12, 13, or 14 different peptides, and mostpreferably 12, 13 or 14 different peptides. Alternatively, a suitablevaccine will preferably contain between 1 and 20 nucleic acid sequences,more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 different nucleic acid sequences, further preferred 6, 7,8, 9, 10 11, 12, 13, or 14 different nucleic acid sequences, and mostpreferably 12, 13 or 14 different nucleic acid sequences.

Such a vaccine is used for active immunization of a mammal, for example,a human who risks being exposed to one or more Plasmodium antigens (forexample, due to travel within a region in which malaria is prevalent).For example, the vaccine can contain at least one antigen selected fromthe group consisting of: 1) a P. falciparum antigen comprising apolypeptide having at least 70% sequence identity with an amino acidsequence selected from the group consisting of an amino acid sequence ofSEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31,34, 35, 28, 39, 42, 43, 46, 47, 66, 67, 70, 72, 74, and/or 76,preferably SEQ ID NO:2, SEQ ID NO:3, or amino acid residues 811-1083 ofSEQ ID NO:3; 2) a P. falciparum antigen comprising a polypeptide havingat least 70% to 99%, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and 99% sequence identity to an amino acid sequenceselected from the group consisting of an amino acid sequence of SEQ IDNOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35,28, 39, 42, 43, 46, 47, 66, 67, 70, 72, 74, and/or 76, preferably SEQ IDNO:2, SEQ ID NO:3, or amino acid residues 811-1083 of SEQ ID NO:3, orfragment thereof; 3) a P. falciparum antigen comprising a polypeptideconsisting essentially of the amino acid sequences of SEQ ID NOs: 2, 3,6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 28, 39,42, 43, 46, 47, 66, 67, 70, 72, 74, and/or 76, preferably SEQ ID NO:2,SEQ ID NO:3, or amino acid residues 811-1083 of SEQ ID NO:3; 4) a P.falciparum antigen consisting of the amino acid sequences of SEQ ID NOs:2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 28,39, 42, 43, 46, 47, 66, 67, 70, 72, 74, and/or 76, preferably SEQ IDNO:2, SEQ ID NO:3, or amino acid residues 811-1083 of SEQ ID NO:3; 5) anucleic acid sequence having at least 70% sequence identity with anucleic acid sequence encoding any one of the peptides listed above,preferably SEQ ID NO: 1 or SEQ ID NO: 4; 6) a nucleic acid sequencehaving at least 70% to 99%, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% and 99% sequence identity to a nucleic acid sequenceencoding the listed polypeptides, preferably SEQ ID NO: 1 or SEQ ID NO:4; 7) a nucleic acid sequence consisting essentially of the nucleic acidsequence sequences described above. and 8) a nucleic acid sequencedescribed above, preferably SEQ ID NO: 1 or SEQ ID NO: 4. A fragment ofthese polypeptides can be approximately 8-56 amino acid residues, suchas 8, 9, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, and 56 residues. Afragment of these nucleic acid sequences can be approximately 10-300nucleotides, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, or 300 nucleotides.

Alternatively, if passive immunization is desired, one can administerone or more antibodies to the following antigens (as a vaccination): 1)a polypeptide having at least 70% sequence identity with an amino acidsequence selected from the group consisting of an amino acid sequence ofSEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31,34, 35, 38, 39, 42, 43, 46, or 47 preferably SEQ ID NO:2, SEQ ID NO:3,or amino acid residues 811-1083 of SEQ ID NO:3; 2) a polypeptide havingat least 70% to 99%, such as 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and 99% sequence identity to an amino acid sequenceselected from the group consisting of an amino acid sequence of SEQ IDNOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35,38, 39, 42, 43, 46, or 47, preferably SEQ ID NO:2, SEQ ID NO:3, andamino acid residues 811-1083 of SEQ ID NO:3, or fragment thereof; 3) apolypeptide consisting essentially of the amino acid sequences of SEQ IDNOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35,38, 39, 42, 43, 46, or 47, preferably SEQ ID NO:2, SEQ ID NO:3, or aminoacid residues 811-1083 of SEQ ID NO:3; and 4) an amino acid sequences ofSEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31,34, 35, 38, 39, 42, 43, 46, or 47, preferably SEQ ID NO:2, SEQ ID NO:3,or amino acid residues 811-1083 of SEQ ID NO:3. A fragment of thesepolypeptides can be approximately 8-56 amino acid residues, such as 8,9, 10, 20, 30, 40, 50, 51, 52, 53, 54, 55, and 56 residues.

The vaccine composition can further comprise an adjuvant and/or acarrier. Examples of useful adjuvants and carriers are given hereinbelow. The peptides and/or polypeptides in the composition can beassociated with a carrier such as e.g. a protein or anantigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into the vaccine compositionincreases or otherwise modifies the immune response to the mutantpeptide. Carriers are scaffold structures, for example a polypeptide ora polysaccharide, to which the neoantigenic peptides, is capable ofbeing associated. Optionally, adjuvants are conjugated covalently ornon-covalently to the peptides or polypeptides of the invention.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedreaction, or reduction in disease symptoms. For example, an increase inhumoral immunity is typically manifested by a significant increase inthe titer of antibodies raised to the antigen, and an increase in T-cellactivity is typically manifested in increased cell proliferation, orcellular cytotoxicity, or cytokine secretion. An adjuvant may also alteran immune response, for example, by changing a primarily humoral or Thresponse into a primarily cellular, or Th response.

Suitable adjuvants include, but are not limited to aluminium salts,Montanide ISA 206, Montanide ISA 50V, Montanide ISA 50, MontanideISA-51, Montanide ISA-720, 1018 ISS, Amplivax, AS15, BCG, CP-870,893,CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, ISPatch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipidA, Montanide IMS 1312, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®vector system, PLG microparticles, resiquimod, SRL172, Virosomes andother Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA)which is derived from saponin, mycobacterial extracts and syntheticbacterial cell wall mimics, and other proprietary adjuvants such asRibi's Detox. Quil or Superfos. Adjuvants such as incomplete Freund's orGM-CSF are preferred. Several immunological adjuvants (e.g., MF59)specific for dendritic cells and their preparation have been describedpreviously (Dupuis M, et al., Cell Immunol. 1998; 186(1):18-27; AllisonA C; Dev Biol Stand. 1998; 92:3-11). Also cytokines may be used. Severalcytokines have been directly linked to influencing dendritic cellmigration to lymphoid tissues (e.g., TNF-alpha), accelerating thematuration of dendritic cells into efficient antigen-presenting cellsfor T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I,et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).

Other examples of useful immunostimulatory agents include, but are notlimited to, Toll-like Receptor (TLR) agonists such as chemicallymodified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U), non-CpGbacterial DNA or RNA as well as immunoactive small molecules, such ascyclophosphamide, which may act therapeutically and/or as an adjuvant.The amounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan without undue experimentation. Additional adjuvantsinclude colony-stimulating factors, such as Granulocyte MacrophageColony Stimulating Factor (GM-CSF, sargramostim). The vaccine may alsocontain a blocker of PD-L1 (CD274) binding to its receptor (PD-1) or toCD80 to prevent/inhibit the development of T regulatory cells (Treg) andthereby reducing the development of tolerance to the vaccine antigen.And exemplary PD-1 inhibitor is Bristol Meyers Squibb's BMS-936558 (alsoknown as MDX-1106 and ONO-4538).

A vaccine composition according to the present invention may comprisemore than one different adjuvants. Furthermore, the inventionencompasses a therapeutic composition comprising any adjuvant substanceincluding any of the above or combinations thereof. It is alsocontemplated that the peptide or polypeptide, and the adjuvant can beadministered separately in any appropriate sequence.

A carrier may be present independently of an adjuvant. The function of acarrier can for example be to increase the molecular weight of inparticular mutant in order to increase their activity or immunogenicity,to confer stability, to increase the biological activity, or to increaseserum half-life. Furthermore, a carrier may aid presenting peptides toT-cells. The carrier may be any suitable carrier known to the personskilled in the art, for example a protein or an antigen presenting cell.A carrier protein could be but is not limited to keyhole limpethemocyanin, serum proteins such as transferrin, bovine serum albumin,human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, orhormones, such as insulin or palmitic acid. For immunization of humans,the carrier must be a physiologically acceptable carrier acceptable tohumans and safe. However, tetanus toxoid and/or diptheria toxoid aresuitable carriers in one embodiment of the invention. Alternatively, thecarrier may be dextrans for example sepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is only possible if atrimeric complex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodimentsthe vaccine composition according to the present invention additionallycontains at least one antigen presenting cell.

In the case of a DNA vaccine, a nucleic acid comprising the sequence ofSEQ ID NOs: 1, 4, 5, 8, 9, 12, 13, 16, 17, 20, 21, 24, 25, 28, 29, 32,33, 36, 37, 40, 41, 44, 45, or 48, preferably SEQ ID NO: 1 or SEQ ID NO:4 formulated in a eukaryotic vector for use as a vaccine that isadministered to human subjects. The nucleotides encoding the antigen areoperably linked promoter and other regulatory sequences in the vector.Such eukaryotic, e.g., mammalian vectors, are known in the art [e.g.,pcDNA™ (Invitrogen) and vectors available from Vical Inc. (San Diego,Calif.)]. Other exemplary vectors, e.g., pNGVL4a, and derivativesthereof, are described in Moorty et al., 2003, Vaccine 21:1995-2002;Cebere et al., 2006, Vaccine 24:41-425; or Trimble et al., 2009, Clin.Cancer Res. 15:364-367; hereby incorporated by reference).

Recombinant Expression Vectors and Host Cells

The antigen of the present invention can be made by any recombinantmethod that provides the epitope of interest. Accordingly, anotheraspect of the invention pertains to vectors, preferably expressionvectors, containing a nucleic acid encoding any clones of Table 1, suchas a PF10_0212a or clone 2 protein, or derivatives, fragments, analogsor homologs thereof. As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a linear or circular double stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.Additionally, some viral vectors are capable of targeting a particularcells type either specifically or non-specifically.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODSIN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectors ofthe invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein (e.g., PF10_0212a or clone 2 proteins,mutant forms of PF10_0212a or clone 2 (e.g., PfSEP-1A, SEQ ID NO:2),fusion proteins, etc.).

The recombinant expression vectors of the invention can be designed forexpression of any of the polypeptides or polynucleotide sequences of thepresent invention in prokaryotic or eukaryotic cells. For example, anyof the polypeptides or polynucleotide sequences of the present inventioncan be expressed in bacterial cells such as E. coli, insect cells (usingbaculovirus expression vectors) yeast cells or mammalian cells. Suitablehost cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example using T7 promoter regulatory sequencesand T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: (1) to increase expression ofrecombinant protein; (2) to increase the solubility of the recombinantprotein; and (3) to aid in the purification of the recombinant proteinby acting as a ligand in affinity purification. Often, in fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the fusion moiety and the recombinant protein to enableseparation of the recombinant protein from the fusion moiety subsequentto purification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31 40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuseglutathione S transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301 315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60 89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, Gottesman, GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 119 128. Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111 2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J 6:229 234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933 943), pJRY88 (Schultz et al.,(1987) Gene 54:113 123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, any of the polypeptides or polynucleotide sequences ofthe present invention can be expressed in insect cells using baculovirusexpression vectors. Baculovirus vectors available for expression ofproteins in cultured insect cells (e.g., SF9 cells) include the pAcseries (Smith et al. (1983) Mol Cell Biol 3:2156 2165) and the pVLseries (Lucklow and Summers (1989) Virology 170:31 39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840)and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187 195). When used inmammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 ofSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue specific regulatory elements are usedto express the nucleic acid). Tissue specific regulatory elements areknown in the art. Non limiting examples of suitable tissue specificpromoters include the albumin promoter (liver specific; Pinkert et al.(1987) Genes Dev 1:268 277), lymphoid specific promoters (Calame andEaton (1988) Adv Immunol 43:235 275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729 733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729 740; Queen andBaltimore (1983) Cell 33:741 748), neuron specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473 5477),pancreas specific promoters (Edlund et al. (1985) Science 230:912 916),and mammary gland specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally regulated promoters are also encompassed, e.g., themurine hox promoters (Kessel and Gruss (1990) Science 249:374 379) andthe α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner that allows forexpression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to mRNA of any of the polynucleotide sequences of thepresent invention. Regulatory sequences operatively linked to a nucleicacid cloned in the antisense orientation can be chosen that direct thecontinuous expression of the antisense RNA molecule in a variety of celltypes, for instance viral promoters and/or enhancers, or regulatorysequences can be chosen that direct constitutive, tissue specific orcell type specific expression of antisense RNA. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus in which antisense nucleic acids are produced under thecontrol of a high efficiency regulatory region, the activity of whichcan be determined by the cell type into which the vector is introduced.For a discussion of the regulation of gene expression using antisensegenes see Weintraub et al., “Antisense RNA as a molecular tool forgenetic analysis,” Reviews Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but also to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, anyof the polypeptides or polynucleotide sequences of the present inventioncan be expressed in bacterial cells such as E. coli, insect cells, yeastor mammalian cells (such as Chinese hamster ovary cells (CHO) or COScells). Alternatively, a host cell can be a premature mammalian cell,i.e., pluripotent stem cell. A host cell can also be derived from otherhuman tissue. Other suitable host cells are known to those skilled inthe art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation, transduction, infection or transfectiontechniques. As used herein, the terms “transformation” “transduction”,“infection” and “transfection” are intended to refer to a variety of artrecognized techniques for introducing foreign nucleic acid (e.g., DNA)into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE dextran mediated transfection, lipofection, orelectroporation. In addition transfection can be mediated by atransfection agent. By “transfection agent” is meant to include anycompound that mediates incorporation of DNA in the host cell, e.g.,liposome. Suitable methods for transforming or transfecting host cellscan be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORYMANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratorymanuals. Transfection may be “stable” (i.e. integration of the foreignDNA into the host genome) or “transient” (i.e., DNA is episomallyexpressed in the host cells).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome theremainder of the DNA remains episomal. In order to identify and selectthese integrants, a gene that encodes a selectable marker (e.g.,resistance to antibiotics) is generally introduced into the host cellsalong with the gene of interest. Various selectable markers includethose that confer resistance to drugs, such as G418, hygromycin andmethotrexate. Nucleic acid encoding a selectable marker can beintroduced into a host cell on the same vector as that encoding any ofthe polypeptides or polynucleotide sequences of the present inventioncan be introduced on a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die). In a specific embodiment, the promoter isthe insulin promoter driving the expression of green fluorescent protein(GFP).

In one embodiment nucleic acid of any of the polypeptides orpolynucleotide sequences of the present invention is present in a viralvector. In another embodiment the nucleic acid is encapsulated in avirus. In some embodiments the virus preferably infects pluripotentcells of various tissue types, e.g. hematopoietic stem, cells, neuronalstem cells, hepatic stem cells or embryonic stem cells, preferably thevirus is hepatropic. By “hepatotropic” it is meant that the virus hasthe capacity to preferably target the cells of the liver eitherspecifically or non-specifically. In further embodiments the virus is amodulated hepatitis virus, SV-40, or Epstein-Bar virus. In yet anotherembodiment, the virus is an adenovirus.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

A transgenic mammal can also be used in order to express the protein ofinterest encoded by one or both of the above-described nucleic acidsequences. More specifically, once the above-described construct iscreated, it can be inserted into the pronucleus of an embryo. The embryocan then be implanted into a recipient female. Alternatively, a nucleartransfer method could also be utilized (Schnieke et al., 1997).Gestation and birth are then permitted to occur (see, e.g., U.S. Pat.Nos. 5,750,176 and 5,700,671), and milk, tissue or other fluid samplesfrom the offspring should then contain the protein of interest. Themammal utilized as the host can be selected from the group consistingof, for example, a mouse, a rat, a rabbit, a pig, a goat, a sheep, ahorse and a cow. However, any mammal can be used provided it has theability to incorporate DNA encoding the protein of interest into itsgenome.

Therapeutic Methods

The invention further provides a method of inducing a P. falciparumspecific immune response in a subject, vaccinating against malaria,treating and or alleviating a symptom of malaria in a subject byadministering the subject a peptide or vaccine composition of theinvention.

The subject has been diagnosed with malaria or is at risk of developingmalaria. The subject has resistant malaria. The subject is a human, dog,cat, horse or any animal in which a P. falciparum specific immuneresponse is desired. Preferably, the subject is a child under 5 yearsold of age. More preferably, the subject is at least about 6-8 weeks oldof age.

The peptide or composition of the invention is administered in an amountsufficient to induce an immune response.

The invention provides methods of treating or prevention malaria byadministering to a subject one or more peptides of the instantinvention. The antigen peptide, polypeptide, nucleic acid sequences orvaccine composition of the invention can be administered alone or incombination with one or more therapeutic agents. The therapeutic agentis, for example, one, two, three, four, or more additional vaccines, anantimalarials artemisinin-combination therapy, or an immunotherapy. Anysuitable therapeutic treatment for malaria may be administered. Theadditional vaccine may comprise an inhibitor of parasite liver invasionor an inhibitor of parasite RBC invasion. Such additional vaccinesinclude, but are not limited to, anti-RBC invasion vaccines (MSP-1),RTS,S (Mosquirix), NYVAC-Pf7, CSP, and [NANP]19-5.1. The antigenpeptide, polypeptide, nucleic acid sequences, or vaccine composition ofthe invention can be administered prior to, concurrently, or after othertherapeutic agents.

The optimum amount of each peptide to be included in the vaccinecomposition and the optimum dosing regimen can be determined by oneskilled in the art without undue experimentation. For example, thepeptide or its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. For example, doses of between 1 and 500 mg 50 μg and 1.5mg, preferably 125 μg to 500 μg, of peptide or DNA may be given and willdepend from the respective peptide or DNA. Doses of this range weresuccessfully used in previous trials (Brunsvig P F, et al., CancerImmunol Immunother. 2006; 55(12):1553-1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017). Other methods of administration of thevaccine composition are known to those skilled in the art.

Pharmaceutical compositions comprising the peptide of the invention maybe administered to an individual already suffering from malaria. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective immune response to thepresent antigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range for the initial immunization(that is for therapeutic or prophylactic administration) from about 1.0μg to about 50,000 μg of peptide for a 70 kg patient, followed byboosting dosages or from about 1.0 μg to about 10,000 μg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient's response and condition by measuring specific immune activityin the patient's blood.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. Preferably, the pharmaceutical compositions areadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. Preferably, the vaccine isadministered intramuscularly. The invention provides compositions forparenteral administration which comprise a solution of the peptides andvaccine compositions are dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine,hyaluronic acid and the like. These compositions may be sterilized byconventional, well known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

The peptide of the invention may also be administered via liposomes,which target the peptides to a particular cells tissue, such as lymphoidtissue. Liposomes are also useful in increasing the half-life of thepeptides. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations the peptide to be delivered isincorporated as part of a liposome, alone or in conjunction with amolecule which binds to, e.g., a receptor prevalent among lymphoidcells, such as monoclonal antibodies which bind to the CD45 antigen, orwith other therapeutic or immunogenic compositions. Thus, liposomesfilled with a desired peptide of the invention can be directed to thesite of lymphoid cells, where the liposomes then deliver the selectedtherapeutic/immunogenic peptide compositions. Liposomes for use in theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9; 467 (1980), USA U.S. Pat. Nos. 4,235,871,4,501,728USA 4,501,728, 4,837,028, and 5,019,369.

For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension containing a peptide may be administered intravenously,locally, topically, etc. in a dose which varies according to, interalia, the manner of administration, the peptide being delivered, and thestage of the disease being treated.

For solid compositions, conventional or nanoparticle nontoxic solidcarriers may be used which include, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.For oral administration, a pharmaceutically acceptable nontoxiccomposition is formed by incorporating any of the normally employedexcipients, such as those carriers previously listed, and generally10-95% of active ingredient, that is, one or more peptides of theinvention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included asdesired, as with, e.g., lecithin for intranasal delivery.

For therapeutic or immunization purposes, nucleic acids encoding thepeptide of the invention and optionally one or more of the peptidesdescribed herein can also be administered to the patient. A number ofmethods are conveniently used to deliver the nucleic acids to thepatient. For instance, the nucleic acid can be delivered directly, as“naked DNA”. This approach is described, for instance, in Wolff et al.,Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and5,589,466. The nucleic acids can also be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253.Particles comprised solely of DNA can be administered. Alternatively,DNA can be adhered to particles, such as gold particles.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in 9618372WOAWO 96/18372; 9324640WOAWO93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);U.S. Pat. Nos. 5,279,833USARose 5,279,833; 9,106,309WOAWO 91/06309; andFelgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

The peptides and polypeptides of the invention can also be expressed byattenuated viral hosts, such as vaccinia or fowlpox. This approachinvolves the use of vaccinia virus as a vector to express nucleotidesequences that encode the peptide of the invention. Upon introductioninto an acutely or chronically infected host or into a noninfected host,the recombinant vaccinia virus expresses the immunogenic peptide, andthereby elicits a host CTL response. Vaccinia vectors and methods usefulin immunization protocols are described in, e.g., U.S. Pat. No.4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectorsare described in Stover et al. (Nature 351:456-460 (1991)). A widevariety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

A preferred means of administering nucleic acids encoding the peptide ofthe invention uses minigene constructs encoding multiple epitopes. Tocreate a DNA sequence encoding the selected CTL epitopes (minigene) forexpression in human cells, the amino acid sequences of the epitopes arereverse translated. A human codon usage table is used to guide the codonchoice for each amino acid. These epitope-encoding DNA sequences aredirectly adjoined, creating a continuous polypeptide sequence. Tooptimize expression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencethat could be reverse translated and included in the minigene sequenceinclude: helper T lymphocyte, epitopes, a leader (signal) sequence, andan endoplasmic reticulum retention signal. In addition, MHC presentationof CTL epitopes may be improved by including synthetic (e.g.poly-alanine) or naturally-occurring flanking sequences adjacent to theCTL epitopes.

The dosing regimen that can be used in the methods of the inventionincludes, but is not limited to, daily, three times weekly(intermittent), two times weekly, weekly, or every 14 days.Alternatively, dosing regimen includes, but is not limited to, monthlydosing or dosing every 6-8 weeks. The vaccine of the present inventioncan be administered intramuscularly once every two weeks for 1, 2, 3, 4,5, or more times, alone or in combination with 1, 2, 3, 4, or moreadditional vaccines in a subject, preferably a human subject.

Antibodies

“Antibody” (Ab) comprises single Abs directed against a target antigen(an anti-target antigen Ab), anti-target antigen Ab compositions withpoly-epitope specificity, single chain anti-target antigen Abs, andfragments of anti-target antigen Abs. A “monoclonal antibody” (mAb) isobtained from a population of substantially homogeneous Abs, i.e., theindividual Abs comprising the population are identical except forpossible naturally-occurring mutations that can be present in minoramounts. Exemplary Abs include polyclonal (pAb), monoclonal (mAb),humanized, bi-specific (bsAb), and heteroconjugate Abs. The inventionencompasses not only an intact monoclonal antibody, but also animmunologically-active antibody fragment, e.g., a Fab or (Fab)2fragment; an engineered single chain Fv molecule; or a chimericmolecule, e.g., an antibody which contains the binding specificity ofone antibody, e.g., of murine origin, and the remaining portions ofanother antibody, e.g., of human origin.

Also provided herein are antibodies to the following antigens (as avaccination): 1) a polypeptide having at least 70% sequence identitywith an amino acid sequence selected from the group consisting of anamino acid sequence of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19,22, 23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or 47, preferablySEQ ID NO:2, SEQ ID NO:3, or amino acid residues 811-1083 of SEQ IDNO:3; 2) a polypeptide having at least 70% to 99%, such as 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity toan amino acid sequence selected from the group consisting of an aminoacid sequence of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23,26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or 47, preferably SEQ IDNO:2, SEQ ID NO:3, or amino acid residues 811-1083 of SEQ ID NO:3, orfragment thereof; 3) a polypeptide consisting essentially of the aminoacid sequences of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22,23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 6, or 47, preferably SEQ IDNO:2, SEQ ID NO:3, or amino acid residues 811-1083 of SEQ ID NO:3; and4) an amino acid sequences of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15,18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or 47,preferably SEQ ID NO:2, SEQ ID NO:3, or amino acid residues 811-1083 ofSEQ ID NO:3. A fragment of these polypeptides can be approximately 8-56amino acid residues, such as 8, 9, 10, 20, 30, 40, 50, 51, 52, 53, 54,55, and 56 residues.

Polyclonal Abs can be raised in a mammalian host by one or moreinjections of an immunogen and, if desired, an adjuvant. Monoclonalantibodies of the invention can be produced by any hybridoma liable tobe formed according to classical methods from splenic or lymph nodecells of an animal, particularly from a mouse or rat, immunized againstthe clone 2 polypeptides or peptides according to the invention.

The antigen and antibody of the present invention can be attached to asignal generating compound or “label”. This signal generating compoundor label is in itself detectable or can be reacted with one or moreadditional compounds to generate a detectable product. Examples of suchsignal generating compounds include chromogens, radioisotopes (e.g.,¹²⁵I, ¹³¹I, ³²P, ³H ³⁵S, and ¹⁴C), fluorescent compounds (e.g.,fluorescein, rhodamine), chemiluminescent compounds, particles (visibleor fluorescent), nucleic acids, complexing agents, or catalysts such asenzymes (e.g., alkaline phosphatase, acid phosphatase, horseradishperoxidase, β-galactosidase, and ribonuclease). In the case of enzymeuse, addition of chromo-, fluoro-, or lumo-genic substrate results ingeneration of a detectable signal. Other detection systems such astime-resolved fluorescence, internal-reflection fluorescence,amplification (e.g., polymerase chain reaction) and Raman spectroscopyare also useful.

Also provided herein is a method of treating P. falciparum malaria in asubject in need of by administering a therapeutically effective amountof an antibody described herewith to the subject. Preferably, theantibody is a purified monoclonal antibody, e.g., one that has beenraised to and is specific for the protein of SEQ ID NO: 2. For example,the monoclonal antibody is a humanized antibody. The treatment can beinitiated at an early stage after the appearance of recrudescentparasites. The symptoms of the subject may be mild or absent andparasitemia is low but increasing, for example from range4,000-10,000/ul. Alternative, the subject may have fever <38.5° C.without any other accompanying symptom. The subject can be a child under10 years of age. The subject can also be an elder child or an adult. Inone example, the subject is characterized as suffering from acute P.falciparum malaria but has not responded to treatment with anti-malarialdrugs. In this passive immunity approach, the purified humanizedmonoclonal antibody that binds specifically to the protein of clones ofTable 1, preferably SEQ ID NO: 2 is administered to the subject to killthe infective agent and/or inhibit RBC invasion.

The antibody can be administered orally, parenterally,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery by catheter or stent, subcutaneously, intraadiposally,intraarticularly, intrathecally, or in a slow release dosage form.Preferably, the antibody is administered intravenously orintramuscularly. For example, the antibody is administered in 1-2 gramamounts, 1, 2, 3, or 4 times. The dosing regimen that can be used in themethods of the invention includes, but is not limited to, daily, threetimes weekly (intermittent), two times weekly, weekly, or every 14 days.Alternatively, dosing regimen includes, but is not limited to, monthlydosing or dosing every 6-8 weeks. The antibody of the present inventioncan be administered intravenously once, twice or three times alone or incombination with 1, 2, 3, 4, or more additional therapeutic agents in asubject, preferably a human subject. The additional therapeutic agentis, for example, one, two, three, four, or more additional vaccines orantibodies, an antimalarials artemisinin-combination therapy, or animmunotherapy. Any suitable therapeutic treatment for malaria may beadministered. The additional vaccine may comprise an inhibitor ofparasite liver invasion or an inhibitor of parasite RBC invasion. Suchadditional vaccines include, but are not limited to, anti-RBC invasionvaccines (MSP-1), RTS,S (Mosquirix), NYVAC-Pf7, CSP, and [NANP]19-5.1.The antibody of the invention can be administered prior to,concurrently, or after other therapeutic agents.

Amounts effective for this use will depend on, e.g., the antibodycomposition, the manner of administration, the stage and severity of P.falciparum malaria being treated, the weight and general state of healthof the patient, and the judgment of the prescribing physician, butgenerally range for the treatment from about 10 mg/kg (weight of asubject) to 300 mg/kg, preferably 20 mg/kg-200 mg/kg.

Kits

Kits are also included within the scope of the present invention. Thepresent invention includes kits for determining the presence ofantibodies to P. falciparum in a test sample. A kit can comprise: (a) aP. falciparum antigen comprising a polypeptide having at least 70%sequence identity with an amino acid sequence selected from the groupconsisting of an amino acid sequence of SEQ ID NOs: 2, 3, 6, 7, 10, 11,14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or47, preferably SEQ ID NO:2, SEQ ID NO:3, or amino acid residues 811-1083of SEQ ID NO:3; and (b) a conjugate comprising an antibody attached to asignal-generating compound capable of generating a detectable signal.The kit can also contain a control or calibrator which comprises areagent which binds to the antigen. The P. falciparum antigen cancomprise a polypeptide having at least 70% to 99%, such as 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity toan amino acid sequence selected from the group consisting of an aminoacid sequence of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18, 19, 22, 23,26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or 47 preferably SEQ IDNO:2, SEQ ID NO:3, and amino acid residues 811-1083 of SEQ ID NO:3, orfragment thereof. A fragment of these polypeptides can be approximately8-56 amino acid residues, such as 8, 9, 10, 20, 30, 40, 50, 51, 52, 53,54, 55, and 56 residues. The antigen can comprise a polypeptideconsisting essentially of the amino acid sequences of SEQ ID NOs: 2, 3,6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39,42, 43, 46, or 47, preferably SEQ ID NO:2, SEQ ID NO:3, or amino acidresidues 811-1083 of SEQ ID NO:3. Finally, the antigen can consist ofthe amino acid sequences of SEQ ID NOs: 2, 3, 6, 7, 10, 11, 14, 15, 18,19, 22, 23, 26, 27, 30, 31, 34, 35, 38, 39, 42, 43, 46, or 47,preferably SEQ ID NO:2, SEQ ID NO:3, or amino acid residues 811-1083 ofSEQ ID NO:3.

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with the vaccine in a formsuitable for intramuscular administration or other routes ofadministration. The kits of the present invention may also contain oneor more antibodies described herewith. Optionally the kit may containdisposable items, such as biodegradable items. The kit may also containa sample collection means, including, but not limited to a needle forcollecting blood, storage means for storing the collected sample, andfor shipment. Alternatively, any kits of the present invention maycontain an instruction for use to diagnose malaria or a receptacle forreceiving subject derived bodily fluid or tissue.

The kit further comprises instructions for use or a CD, or CD-ROM withinstructions on how to collect sample, ship sample, and means tointerpret test results. The kit may also contain a control sample eitherpositive or negative or a standard and/or an algorithmic device forassessing the results and additional reagents and components.

A “biological sample” is any bodily fluid or tissue sample obtained froma subject, including, but is not limited to, blood, blood serum, urine,and saliva.

The kit may further comprise one or more additional compounds togenerate a detectable product. Examples of such signal generatingcompounds include chromogens, radioisotopes (e.g., ¹²⁵I, ¹³¹I, ³²P, ³H,³⁵S, and ¹⁴C), fluorescent compounds (e.g., fluorescein, rhodamine),chemiluminescent compounds, particles (visible or fluorescent), nucleicacids, complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase, β-galactosidase,and ribonuclease).

By way of example, and not of limitation, examples of the presentinvention shall now be given.

Example 1: Antibodies to PfSEP-1Block Parasite Egress from RBCs andProtect Subjects from Severe Malaria

P. falciparum malaria is a leading cause of morbidity and mortality indeveloping countries, infecting hundreds of millions of individuals andkilling over one million children in sub-Saharan Africa each year.Recent estimates indicate that even these staggering figuressignificantly underestimate the actual disease burden. Children sufferthe greatest morbidity and mortality from malaria-yet this age group hasnot been targeted at the identification stage of vaccine development. Ofthe about 100 vaccine candidates currently under investigation, morethan 60% are based on only four parasite antigens. New antigencandidates are urgently needed, but strategies to identify novelantigens are limited and many focus on rodent malarias.

Human residents of endemic areas develop protective immunity that limitsparasitemia and disease, and naturally acquired human immunity providesan attractive model for vaccine antigen identification. Plasma from somechronically exposed individuals contains antibodies which limit parasitegrowth ex vivo and following adoptive transfer, a finding which confirmsthe protective efficacy of anti-parasite antibodies. One approach toidentify and characterize new malarial vaccine candidate antigens is toidentify malarial proteins that are uniquely recognized by antibodies inthe plasma of chronically exposed, yet resistant individuals. Because oflogistic difficulties in characterizing naturally acquired resistance inendemic populations, this approach has not been widely exploited.

Studies were carried out to identify vaccine candidates for pediatricfalciparum malaria by identifying the parasite targets of naturallyacquired protective human antibodies. A differential, whole proteomescreening method using plasma and epidemiologic data from a birth cohortof children living in Tanzania was used to identify P. falciparumantigens associated with resistance in two-year old children. SchizontEgress Protein-1 (PfSEP-1), a 244-kDa parasite antigen, which localizesto the schizont/parasitophorous vacuole membrane, Maurer's clefts andthe inner leaflet of the RBC membrane was identified in schizontinfected RBCs. Antibodies to PfSEP-1 decrease parasite replication by60% by arresting schizont rupture. Active vaccination with rPbSEP-1resulted in a 4.5 fold reduction in parasitemia after challenge with P.berghei ANKA parasites. Children in the cohort experienced adramatically increased incidence of severe malaria during periods withundetectable anti-PfSEP-1 antibody levels (45 cases/23,806 child weeks)compared to periods with detectable antibody levels (0 cases/1,688 childweeks). By blocking schizont egress, PfSEP-1 synergizes with vaccinestargeting hepatocyte and red cell invasion.

Identification and In Vitro Evaluation of Vaccine Candidates

Using a differential screening method, the P. falciparum blood stageproteome with plasma from resistant and susceptible two yr old childrenwas interrogated to identify parasite proteins that are the targets ofprotective antibody responses. We focused on 2 yr old children becausein our cohort, resistance to parasitemia is first detected at this age(FIG. 6). We selected twelve resistant and eleven susceptible 2 year oldchildren with careful matching for potential non-immunologic factors,which may be related to resistance (see Table below and FIG. 16).Resistance was determined based on the geometric mean parasite densityon all blood films collected between ages 2 and 3.5 yrs. We pooledplasma collected at age 2 yrs (+/−2 weeks) from the resistantindividuals (RP) and susceptible individuals (SP) and performeddifferential screening experiments on a P. falciparum 3D7 strain bloodstage cDNA library. We screened 1.25×10⁶ clones and identified threeclones that were uniquely recognized by RP, but not SP. The sequences ofthese clones were compared to the published falciparum genome(PlasmoDB.org) and found to encode nt 2,431-3,249 of PF3D7_1021800—agene on chromosome 10, nt 3,490-5,412 of PF3D7 1134300—a gene onchromosome 11, and nt 201-1,052 of PF3D7 1335100—which encodes merozoitesurface protein-7 (MSP-7)—a protein involved in RBC invasion which iscurrently under study as a potential vaccine candidate.

In silico analysis (PlasmoDB.org) predicts that PF3D7_1021800 contains a6225 bp gene that encodes a 244-kDa acidic phospho-protein (SEQ IDNO:2), contains two introns near its 3′ end, and has syntenic orthologsin all rodent and human malarias evaluated. Based on in vitroexperiments, we designate the protein product of PF3D7_1021800 asPlasmodium falciparum Schizont Egress Protein 1 (PfSEP-1). PF3D7_1021800mRNA expression increases throughout blood stage schizogeny and the genedisplays minimal sequence variation, with no SNPs in the cloned region(nt 2,431-3,249), across fifteen field and laboratory isolates (FIG.16). A recently reported deep sequencing effort on 227 field samplesidentified 3 non-synonymous and 1 synonymous SNPs in the cloned region.We have sequenced nt 2,431-3,249 of PF3D7_1021800 in 6 field isolatesobtained from children in our cohort and found one isolate with a six bpinsertion (encoding Asp-Gly-Asp-Gly instead of the canonical Asp-Gly) aswell as one synonymous SNP. These data indicate that there is little orno sequence variability among parasite strains.

PfSEP-1 has no significant homology to proteins of known function. Toexplore the function of PfSEP-1, we have constructed vectors designed todisrupt the coding and promoter regions of the gene through the welldescribed process of homologous recombination⁹. We have obtainedepisomal carriage of both targeting vectors, but have not recoveredhomologous integrants with either vector, suggesting that expression ofPF3D7_1021800 is essential for blood stage replication (FIGS. 8A-C).

We have expressed and purified the polypeptide encoded by nt 2,431-3,249of PF3D7_1021800 (aa 810-1083) in E. coli and designated thisrecombinant protein rPfSEP-1A (FIG. 9). Using an independent selectionof resistant and susceptible individuals (see Table below), we confirmedand generalized the differential recognition of rPfSEP-1A (SEQ ID NO:2)in an ELISA based assay. IgG antibody recognition of rSEP-1A was 4.4fold higher in RP (n=11) than in SP (n=14, P<0.0002, FIG. 10), yet didnot differ for other malarial proteins or controls.

P Variable Resistant Susceptible value^(a) Number of Subjects 12 11 —Hemoglobin phenotype (% AS) 16.6 0 0.47 Sex (% female) 41.6 45.4 1 Weeksof follow-up 140.5 [44.5] 152 [44] 0.31 (median [IQR]) # of Blood smearsfrom age 16.5 [21.5] 21 [24] 0.31 2-3.5 yrs (median [IQR]) # of PositiveBlood smears from 0 [1] 4 [10] 0.04 age 2-3.5 yrs (median [IQR]) # ofanti-malarial treatments 2 [1.75] 8 [8] 0.01 before age 2 yrs (median[IQR]) Pregnancy malaria (%) 16.6 9 1 Maternal age (yrs, median [IQR])22.5 [9.5] 28 [10] 0.35 Birth Season (% in High Season) 25 9 0.59Children using Bed Net (%) 33.3 0 0.09 # of Previous Pregnancies 0 [2] 1[2] 0.19 (median [IQR]) Parasite density (parasites/ 0 [0] 0 [0] 1 200WBCs) at 2 yr blood draw (median [IQR]) Parasite density (parasites/ 0[25.6] 3203 [944.1] 0.05 200 WBCs) from a 2-3.5 yrs (median [IQR])^(a)Comparisons of catagorical variables by 2 tailed Fisher's exacttest. Comparisons of continuous variables by Mann-Whitney U test

P Variable Resistant Susceptible value^(a) Number of Subjects 11 14 1Hemoglobin phenotype (% AS) 36 21 0.66 Sex (% female) 45 43 1 Weeks offollow-up 154 [14] 165 [19] 0.34 (median [IQR]) # of Blood smears fromage 14 [5.8] 20.5 [9.5] 0.02 2-3.5 yrs (median [IQR]) # of PositiveBlood smears from 0 7.8 [6] <0.001 age 2-3.5 yrs (median [IQR]) # ofanti-malarial treatments 2.6 [2.9] 6.3 [3.1] 0.008 before age 2 yrs(median [IQR]) Pregnancy malaria (%) 9 14 1 Maternal age (yrs, median[IQR]) 27 [8] 27 [7] 0.85 Birth Season (% in High Season) 73 50 0.41Children using Bed Net (%) 0 0 1 # of Previous Pregnancies 1 [3.0] 1[3.0] 0.89 (median [IQR]) Parasite density (parasites/ 0 [0] 0 [0] 1 200WBCs) at 2 yr blood draw (median [IQR]) Parasite density (parasites/ 0[0] 2106.9 [2700] <0.001 200 WBCs) from a 2-3.5 yrs (median [IQR])^(a)Comparisons of catagorical variables by 2 tailed Fisher's exacttest. Comparisons of continuous variables by Mann-Whitney U test

We have cloned this sequence into a eukaryotic expression plasmid(VR2001), immunized mice and generated anti-rPfSEP-1A anti-sera. Toconfirm that PF3D7_1021800 encodes a parasite protein, we probed P.falciparum 3D7 infected and uninfected RBCs with both pre-immune andpost-immune sera. Anti-rPfSEP-1A recognized a 244-kDa protein ininfected but not uninfected RBC (FIGS. 11A-B).

We performed growth inhibition assays using anti-rPfSEP-1A antiseraprepared by both DNA vaccination and recombinant protein immunization.Parasites were synchronized to the ring stage, cultured to obtain maturetrophozoites and then incubated with anti-rPfSEP-1A antisera or controlsfor 24 hr followed by enumeration of newly invaded ring stage parasites.Anti-rPfSEP-1A generated by both DNA plasmid and recombinant proteinbased immunization inhibited parasite growth by 58-75% across threeparasite strains compared to controls (all P<0.009). Antisera preparedby DNA vaccination against an irrelevant falciparum protein(phosphatidylglycerophosphate synthase, PF3D7_0820200) showed no growthinhibition.

As shown in FIG. 19, rabbit anti-PfSEP-1 inhibits parasitegrowth/invasion by 68% in vitro. Ring stage 3D7 parasites weresynchronized twice using sorbitol plated at 1% parasitemia, allowed tomature to trophozoites (24 hrs), followed by addition of anti-clone 2rabbit sera (1:10 dilution). Negative controls included no rabbit seraand pre-immune rabbit sera (1:10 dilution). Parasites were cultured for24 hrs and ring stage parasites were enumerated by microscopicexamination. Bars represent the mean of 3 independent replicates. Errorbars represent SEMs. P<0.0001 for comparison between pre and post immunerabbit sera by non-parametric Mann-Whitney U test.

We immunolocalized PfSEP-1 by both immunofluorescence confocalmicroscopy and immunogold transmission electron microscopy (FIGS. 2A-C).Anti-PfSEP-1 did not bind to free merozoites, rings or late trophozoitestage parasites, but did specifically recognize an antigen expressed bylate schizont infected RBC (FIGS. 2A-B). In non-permeabilized, non-fixedschizont infected RBCs, PfSEP-1 co-localized with glycophorin A (FIG.2C). This localization was further evaluated by immunoelectronmicroscopy (FIG. 2D). In non-permeabilized, non-fixed schizont infectedRBCs, PfSEP-1 localized to the schizont/parasitophorous vacuolemembrane, Maurer's clefts and the inner leaflet of the RBC membranewhile glycophorin A was confined to the outer leaflet of the RBCmembrane. This pattern of staining was observed in essentially all ofthe late schizont infected RBCs examined. No staining for PfSEP-1 wasobserved in uninfected RBC or ring/trophozoite infected RBCs (FIGS.13A-B). The close juxtaposition of these structures in late schizontinfected RBCs with the RBC outer membrane explains the apparentco-localization of PfSEP-1 with glycophorin A observed by confocalmicroscopy. The accessibility of antibodies to PfSEP-1 innon-permeabilized, non-fixed schizont infected RBCs is consistent withthe known permeability of parasitized RBCs at the later stages ofschizogony.

The localization of PfSEP-1 was not consistent with a role in RBCinvasion, rather it suggested a role in parasite egress from infectedRBCs. To determine the mechanism of growth inhibition we performedschizont arrest assays using anti-rPfSEP-1A antisera prepared by bothDNA vaccination (FIGS. 3A-C) and recombinant protein immunization (FIGS.14A-B). Parasites were synchronized to the ring stage at high (3.5%)parasite density, cultured to obtain early schizonts and then incubatedwith anti-rPfSEP-1A antisera or controls for 12 hr followed byenumeration of remaining schizont stage parasites. Under theseconditions, the majority of schizont infected RBCs should rupture,releasing merozoites, which would invade new RBCs and develop into ringstage parasites. Anti-rPfSEP-1A generated by both DNA plasmid andrecombinant protein based immunization dramatically inhibited schizontegress resulting in 4.3-6.8 fold higher proportion of schizonts acrossthree parasite strains compared to controls (all P<0.009).

Active Vaccination with SEP-1 Protects Mice from P. berghei Challenge

To evaluate the protective efficacy of active vaccination with SEP-1 invivo, we cloned the P. berghei ANKA strain ortholog of PfSEP-1 (nt2173-3000) into the expression plasmid pET30 and expressed and purifiedrPbSEP-1A (aa 725-1000) from (FIG. 4A). We vaccinated Balb/C mice (n=11)with rPbSEP-1A in TiterMax Gold adjuvant or adjuvant alone (n=11),measured their antibody responses to rPbSEP-1A (FIG. 4B), and challengedthem with 10⁶ P. berghei ANKA parasite infected red blood cellsintraperitoneally. Mice vaccinated with rPbSEP-1A had 4.5 fold decreasedparasitemia on day 7 post challenge compared to controls treated withadjuvant alone (FIG. 4C).

Human Antibody Responses to PfSEP-1

To evaluate the impact of naturally acquired anti-PfSEP-1 antibodies onclinical malaria, we measured anti-PfSEP-1 IgG antibody levels using afluorescent, bead-based assay in our birth cohort and related theselevels to subsequent malaria outcomes. We measured anti-PfSEP-1 IgGantibody levels in available plasma obtained at scheduled, non-sickvisits between 2 and 3.5 yrs of life (total of 156 antibody measures on155 children). Anti-PfSEP-1 antibodies were detectable in 3.2% of thesesamples and children were followed for a total of 6,350 child-weeks ofobservation (201 weeks with detectable anti-PfSEP-1 and 6,149 weeks withundetectable levels). We related the presence of detectable anti-PfSEP-1antibodies to malarial outcomes, including parasite density, mildmalaria, severe malaria, all cause and malaria attributed mortality. Foreach antibody measurement, the time interval examined for malariaoutcomes extended from the time of the antibody measurement until thechild had a subsequent antibody determination or completed the study.

We used generalized estimating equations (GEE) based longitudinalregression models to evaluate the relationship between time varyinganti-PfSEP-1 antibody responses and dichotomous malaria endpoints.Similar GEE based linear regression models were used for the continuousendpoints of parasite density on all available blood smears and parasitedensity on positive blood smears. These models adjust for both potentialconfounders and the lack of independence (correlation) amongobservations taken from the same subject over time. Potentialconfounders included hemoglobin phenotype, age, and average priorparasitemia on all blood smears.

Children without detectable anti-PfSEP-1 IgG antibody had higherparasite densities on all available blood smears, higher parasitedensities on positive blood smears, and increased incidence of mildmalaria. (FIGS. 15A-C).

Severe malaria did not occur during periods when children had detectableanti-PfSEP-1 antibody levels (0 cases/201 child weeks with detectableanti-PfSEP-1 antibody vs. 6 cases/6,149 child weeks with undetectableanti-PfSEP-1 antibody), however the small number of total casesprecluded meaningful analysis. In our cohort, severe malaria is stronglyage dependent with the majority of cases occurring before 2 yrs of age.To increase the number of severe malaria cases for analysis, we extendedthe age range examined to 1.5-3.5 yrs of life encompassing 687 antibodymeasures on 453 children. Anti-PfSEP-1 antibodies were detectable in6.0% of these samples and children were followed for a total of 25,494child-weeks of observation (1,688 child weeks with detectableanti-PfSEP-1 and 23,806 child weeks with undetectable levels).Strikingly, severe malaria did not occur during periods when childrenhad detectable anti-PfSEP-1 antibody levels (0 cases/1,688 child weekswith detectable anti-PfSEP-1 antibody vs. 45 cases/23,806 child weekswith undetectable anti-PfSEP-1 antibody, FIG. 5).

Individuals without detectable anti-PfSEP-1 IgG antibody hadsignificantly increased risk of developing severe clinical malaria(adjusted OR 4.4; Type III fixed effects P<0.01) compared to individualswith detectable anti-PfSEP-1 IgG antibody levels even after adjustingfor potential confounders. There was no significant difference in therisk for all-cause mortality or malaria-associated mortality, though theevent rates for mortality were low. These results represent the firstdemonstration that antibodies that specifically block schizont egresscan protect against severe malaria in humans.

Blocking Parasite Egress Protects Against Malaria

Falciparum malaria remains a leading cause of childhood mortality andvaccines are urgently needed to attenuate this public health threat. Wereport the rational identification of vaccine candidates by identifyingparasite proteins uniquely recognized by antibodies expressed byresistant, but not susceptible children. Using a differential screen, weidentified two genes encoding useful vaccine antigens as well as MSP-7,a known vaccine candidate. We have extensively characterized PfSEP-1,the protein product of PF3D7 1021800. PfSEP-1 localizes to theschizont/parasitophorous vacuole membrane, Maurer's clefts and the innerleaflet of the RBC membrane in schizont infected RBCs. PfSEP-1 isaccessible to antibodies during late schizogeny, and displays minimalsequence variation, particularly in the region identified by ourdifferential screening experiments (aa 810-1083; SEQ ID NO:2).Antibodies to PfSEP-1 significantly attenuate parasite growth via aunique mechanism; arresting schizont egress from infected RBCs withoutcausing schizont agglutination.

Schizont egress is a complex tightly regulated process involving calciumdependent phosphorylation of parasite target proteins followed byproteolytic remodeling of parasite, as well as RBC cytoskeletalproteins. One of these proteolytic events involves SERA-5, the target ofantibodies that agglutinate merozoites and schizonts and mediateschizont killing in cooperation with complement. Unlike SERA 5 and otherproteins involved in schizont egress, PfSEP-1 was not identified inglobal profiles of proteolysis during schizont egress, and we did notobserve any evidence of cleavage events within PfSEP-1 at any bloodstage of development. The localization of PfSEP-1 to the inner RBCleaflet is consistent with a role in remodeling the RBC cytoskeletonprior to rupture.

In active vaccination experiments, rPbSEP-1A conferred marked protectionagainst P. berghei ANKA challenge as evidenced by a 4.5 fold reductionin parasitemia seven days post-challenge. In addition, vaccination withrPbSEP-1A resulted in self-cure in one out of eleven vaccinated mice.These data constitute the first report of protection in P. berghei byvaccines targeting schizont egress and offer a pathway forward foradvancing these vaccines toward non-human primate models.

In our longitudinal birth cohort, anti-PfSEP-1 antibodies wereassociated with significant protection from severe malaria, with nocases occurring while children had detectable anti-PfSEP-1 antibodies.This represents the first time that antibodies that specifically blockschizont egress have been associated with protection from severemalaria. Under conditions of natural exposure, only 6% of 1.5 to 3.5 yrold children in our cohort had detectable anti-PfSEP-1 antibodies. Thislow natural prevalence suggests that adjuvanted vaccination with PfSEP-1could have a marked impact on reducing severe malaria in young children.

The data validate the field-to-lab-to-field based strategy for therational identification of vaccine candidates and indicate that PfSEP-1is useful as a vaccine for pediatric falciparum malaria. By blockingschizont egress, PfSEP-1 synergizes with vaccines targeting hepatocyteand red cell invasion such as MSP-4, MSP-7, and/or RTSS.

The following materials and methods were used to generate the datadescribed herein.

Study Population

Subjects participated in the Mother Offspring Malaria Studies (MOMS)project, which is based at Muheza Designated District Hospital (DDH), innorth eastern Tanzania. Mothers presenting at Muheza DDH for deliverywere enrolled and provided signed, informed consent prior toparticipation of themselves and their newborns in the study. Details ofthe MOMS study design, enrolment methods, and exclusion criteria havebeen described (Mutabingwa et al., PLoS Med 2, e407 (2005), andKabyemela et al., J. Infect. Dis. 198, 163-166 (2008))

Inclusion Criteria and Clinical Monitoring

We monitored N=785 children for P. falciparum infection from birth up to3.5 years of age. Children were evaluated at routine, well-child visitsby a clinician every two weeks from birth to one year of age, andmonthly thereafter, including blood smear analysis. Routine bloodsamples were collected once every 6 months from 1.5 to 3.5 years oflife. Blood smears and blood samples were also collected any time thechild became sick. Sick children were examined by a medical officer uponpresentation to the hospital or mobile clinic. Treatment outside thestudy was minimized by active, weekly surveillance by our mobileclinics.

Clinical malaria was defined as asexual P. falciparum parasitemia byblood smear coupled with symptoms suggestive of malaria such astemperature >37.5° C., nausea or vomiting, irritability, and poorfeeding. Prompt treatment was provided to sick children according to theguidelines of the Tanzanian Ministry of Health, and study participantswere instructed to obtain all medications including antimalarialsthrough the project staff.

Sample Collection and Processing

Venous blood was collected and stored at 4° C. until processing.Following centrifugation, plasma was stored at −80° C. P. falciparumparasitemia was determined by Giemsa-stained thick blood smears preparedfrom capillary or venous blood. Parasite density was expressed as thenumber of asexual stage parasites/200 white blood cells in the thicksmear. Sickle cell trait was determined by electrophoresis (HelenaLaboratories, Beaumont, Tex. USA). Hemograms were obtained on animpedance-based analyzer (Abbott Cell Dyne® 1200).

Case Definitions

Mild malaria was defined as a positive bloodsmear and one or more of thefollowing: 1) anemia defined by Hgb<6 g/dL; 2) vomiting; 3) diarrhealdisease or gastroenteritis; 4) lower respiratory infection; or 5) oraltemperature>=38 deg C.

Severe malaria was defined as a positive bloodsmear and one or more ofthe following: 1) respiratory distress defined by respiratory rateof >40/min for children older than two months of age or a respiratoryrate of >50/min for children less than two months of age; 2) a historyof one or more convulsions in the twenty-four hours prior to or duringhospitalization; 3) prostration defined by inability to sit unaided; 4)hypoglycemia defined by glucose <2.2 mmol/L; 5) severe anemia defined byHgb<6 g/dL; or 6) oral temperature>40 deg C.

Malaria-associated mortality was defined as death with a positive bloodfilm obtained during the terminal illness. One child who died ofbacterial meningitis, but had a positive blood film was adjudicated as anon-malarial death.

Selection of Resistant and Susceptible Individuals

We excluded individuals with less than 9 of the total n=18 scheduledmonthly blood smears collected between the ages of 2-3.5 yrs,individuals with less than 200 ul of plasma available from the plasmasample obtained at age 2 (+/−2 weeks), and individuals who wereparasitemic at the time the 2 yrs (+/−2 weeks) plasma sample wasobtained. We then rank ordered individuals based on the geometric meanparasite density on all blood films collected between ages 2 and 3.5yrs. This mean parasite density included the scheduled monthly bloodsmears as well as positive blood smears obtained during sick visits. Tenindividuals from the high and low extremes of this distribution werechosen to comprise the Resistant and Susceptible groups. Selections weremade with matching based on village of residence, # ofmalaria-associated clinic visits, sex, and # of doses of anti-malarials.Potential confounders examined included: Hgb phenotype, presence ofplacental malaria, maternal age, birth season, use of bed nets, and # ofprevious pregnancies. A second, independent selection of resistant andsusceptible individuals (table S2) was chosen for ELISA-basedconfirmatory assays.

Whole Proteome Differential Screening

We obtained a P. falciparum blood-stage cDNA expression library inLambda Zap (MRA-299) from MR4. We plated this library at 25,000clones/plate on 150 mm NZY plates in XL-1 Blue strain of E. coli.Duplicate IPTG-soaked nitrocellulose filters were prepared from each of50 plates. Filters were blocked in 5% milk, TBS pH 7.4 (MTBS). Resistantplasma (RP) and susceptible plasma (SP) were diluted 1:100 in MTBS.Duplicate filters were probed with either RP or SP for 3 hr at 37 degCelsius. Filters were washed 3×5 min in 0.05% Tween 20, TBS pH 7.4(TTBS) and probed with alkaline phosphatase conjugated anti-human IgGdiluted 1:5000 in MTBS for 1 hr at 37 deg Celsius. Filters were washed3×5 min in TTBS. Filters were developed in BCIP/NBT. Clones whichreacted with RP but not SP were cored out of their corresponding plate,eluted in SM buffer, re-plated and re-screened. Three rounds of plaquepurification typically resulted in homogeneous clones which are reactivewith RP but not reactive with SP. cDNA inserts uniquely reactive with RPwere recovered by PCR amplification using vector specific primers andsequenced.

PfSEP-1A Expression and Purification

We subcloned the ORF encoding as 810-1083 of PfSEP-1 into pET30(Novagen) and transformed the resulting plasmid into the expression hostE. coli BL21(DE3) (Novagen). Transformants were grown in Terrific brothsupplemented with 100 tig/mL kanamycin, at 37 deg C. in a 10 L fermenterwith oxygen sparging (10 L/min) until OD600=8.0.Isopropyl-b-D-thiogalactopyranoside was added to a final concentrationof 1 mmol/L, and the culture was fed continuously with 0.3 g/ml glucose,0.09 g/ml yeast extract at 50 ml/hr for 12 h. Cultures were harvested bycentrifugation and 750 gr of wet cell paste was resuspended in 10 L of10 mmol/L potassium phosphate, 150 mmol/L NaCl, and 10 mmol/L imidazole(pH 8.0) and lysed by high pressure disruption at 20,000 PSI(Microfluidics, Model 110-T). The lysate was clarified by tangentialflow microfiltration (filter area 1m2, pore size 1 um, Milipore) and 8 Lof clarified lysate was recovered.

Protein purification was achieved by a 4-step process on BioPilotchromatography equipment (Pharmacia). Briefly, clarified lysate wasapplied to a FineLine Pilot 35 (GE Healthcare) column containing 90 mLof Ni-NTA Superflow Resin (Novagen). The protein of interest was elutedwith a stepped gradient containing increasing concentrations ofimidazole. Fractions containing the protein of interest were pooled,adjusted to 400 mmol/L ammonium sulfate, 10 mmol/L DTT and furtherpurified, by hydrophobic-interaction chromatography on a FineLine Pilot35 (GE Healthcare) column containing 150 ml of Source 15PHE (GEHealthcare). Recombinant proteins were eluted with a linear gradient ofelution buffer (10 mmol/L Tris, 1 mmole/L DTT, 1 mmol/L EDTA [pH 8.0]).Fractions containing the protein of interest were pooled, and furtherpurified, by anion exchange chromatography on a FineLine Pilot 35 (GEHealthcare) column containing 130 ml of MacroPrep High Q (BioRad).Recombinant proteins were eluted with a linear gradient of elutionbuffer (10 mmol/L Tris, 1 mole/L NaCl, 1 mmole/L DTT, 1 mmol/L EDTA [pH8.0]). Final purification was achieved by ceramic hydroxyapatitechromatography on a FineLine Pilot 35 (GE Healthcare) column containing70 ml of CHT type 1 (BioRad). Recombinant proteins were eluted with alinear gradient of elution buffer (500 mmole/L potassium phosphate, and1 mmole/L DTT, pH 7.4)

Purified rPfSEP-1A was buffer exchanged into 10 mmol/L sodium phosphate,0.05% Tween 20, 3% sucrose and concentrated to 500 μg/ml by tangentialflow ultrafiltration (filter area 50 cm2, pore size 5 kDa, Pall).rPFSEP-1A was lyophilized at 500 μg/vial and stoppered under nitrogen.Endotoxin levels were less than 2EU/mg protein as determined by an FDAcleared assay (Lonza). Typical yields are >50 mg rPfSEP-1A per 750 gr ofwet cell paste.

PbSEP-1A Expression and Purification

We subcloned the ORF encoding as 725-1000 of PbSEP-1 into pET30(Novagen) and transformed the resulting plasmid into the expression hostE. coli BL21(DE3) (Novagen). Transformants were grown in Terrific brothsupplemented with 100 μg/mL kanamycin, at 37 deg C. in a 10 L fermenterwith oxygen sparging (10 L/min) until OD600=8.0.Isopropyl-b-D-thiogalactopyranoside was added to a final concentrationof 1 mmol/L, and the culture was fed continuously with 0.3 g/ml glucose,0.09 g/ml yeast extract at 50 ml/hr for 12 h. Cultures were harvested bycentrifugation and 750 gr of wet cell paste was resuspended in 10 L of10 mmol/L potassium phosphate, 150 mmol/L NaCl, and 10 mmol/L imidazole(pH 8.0) and lysed by high pressure disruption at 20,000 PSI(Microfluidics, Model 110-T). The lysate was clarified by tangentialflow microfiltration (filter area 1m2, pore size 1 um, Milipore) and 8 Lof clarified lysate was recovered.

Protein purification was achieved by a 3-step process on BioPilotchromatography equipment (Pharmacia). Briefly, clarified lysate wasapplied to a FineLine Pilot 35 (GE Healthcare) column containing 90 mLof Ni-NTA Superflow Resin (Novagen). The protein of interest was elutedwith a stepped gradient containing increasing concentrations ofimidazole. Fractions containing the protein of interest were pooled,adjusted to 400 mmol/L ammonium sulfate, 10 mmol/L DTT and furtherpurified, by hydrophobic-interaction chromatography on a FineLine Pilot35 (GE Healthcare) column containing 150 ml of Source 15PHE (GEHealthcare). Recombinant proteins were eluted with a linear gradient ofelution buffer (10 mmol/L Tris, 1 mmole/L DTT, 1 mmol/L EDTA [pH 8.0]).Fractions containing the protein of interest were pooled, and furtherpurified, by anion exchange chromatography on a FineLine Pilot 35 (GEHealthcare) column containing 130 ml of MacroPrep High Q (BioRad).Recombinant proteins were eluted with a linear gradient of elutionbuffer (10 mmol/L Tris, 1 mole/L NaCl, 1 mmole/L DTT, 1 mmol/L EDTA [pH8.0]).

Purified rPbSEP-1A was buffer exchanged into 10 mmol/L sodium phosphate,0.05% Tween 20, 3% sucrose and concentrated to 125 μg/ml by tangentialflow ultrafiltration (filter area 50 cm2, pore size 5 kDa, Pall).rPFSEP-1A was lyophilized at 125 μg/vial and stoppered under nitrogen.Endotoxin levels were less than 2EU/mg protein as determined by an FDAcleared assay (Lonza). Typical yields are >50 mg rPbSEP-1A per 750 gr ofwet cell paste.

Parasite Strains and Culture

P. falciparum strains (3D7, D10, and W2) were obtained from MR4. Theparasites were cultured in vitro according to the methods of Trager andJensen with minor modifications 29. Briefly, parasites were maintainedin RPMI 1640 medium containing 25 mm HEPES, 5% human 0+ erythrocytes,0.5% Albumax II (Invitrogen) or 10% heat inactivated human AB+serum, 24mm sodium bicarbonate, and 10 μg/ml gentamycin at 37° C. with 5% CO2, 1%02, and 94% N2.

P. berghei ANKA was obtained from MR4 as a stabilite and was expanded inBalb/C mice prior to challenge studies.

Anti-PfSEP-1 Antisera Production

Mouse anti-PfSEP-1 antisera was produced by either DNA or recombinantprotein immunization. For DNA immunization, we subcloned the ORFencoding as 810-1083 of PfSEP-1 into VR2001, transformed into the hostE. coli NovaBlue (Novagen), and purified endotoxin free plasmid(Endofree Giga, Qiagen). Balb/C mice were immunized with 180 μg ofplasmid (50 ug intramuscular injection in each hind leg and 80 μgintradermal injection at base of tail) followed by 80 μg intradermalinjections at base of tail every two weeks for a total of four doses.For protein immunization, we emulsified rPfSEP-1 in an equal volume ofTiterMax adjuvant (CytRx Corporation) and injected 50 μg of rPfSEP-1intraperitoneally at two week intervals for a total of four doses.

Western Blot

Parasite pellets were prepared by treatment of parasitized RBCs with0.15% saponin in phosphate buffered saline (PBS), pH 7.4 on ice for 10min followed by centrifugation (3,000×g, 5 min), and resuspension incold PBS, and centrifugation (3,000×g, 5 min). Parasite pellets orrPfSEP-1A were dissolved in SDS sample loading buffer (Bio-Rad), heatedto 95 deg C. for 10 min, and proteins were separated in 4-11% gradientSDS-PAGE gels. Separated proteins were transferred to nitrocellulosemembranes which were blocked in 5% milk PBS (pH 7.4) and 0.05% Tween 20for 1 h. Membranes were probed with polyclonal anti-PfSEP-1A orpre-immune mouse sera, detected by use of anti-mouse IgG antibodyconjugated to alkaline phosphatase, and developed with5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma).

SNP Detection in Field Isolates

We extracted DNA from filter paper containing dried blood spots obtainedfrom six parasitemic children in our cohort (QIAmp DNA Blood Mini Kit,Qiagen). We amplified nt 2,431-3,249 of PF3D7_1021800 from extracted DNAusing a nested PCR based approach. First round primers were: Fl5′-GAAGATGTTTGTCATAATAATAACGTGGAAGACC-3′ (SEQ ID NO: 49), R15′-TCCTACAACATCTATTTCTCCTGTGTAAGG-3′. (SEQ ID NO: 50) Second roundprimers were: F2 5′-GAATAAAAAAATGGATGAGATGAAAG-3′(SEQ ID NO: 51), R25′-CTATTACTATCCTCATTTGCATCTGTATATTTATCC-3′(SEQ ID NO: 52). First roundPCR conditions were: 10 min initial denature at 94 deg C. followed by 40cycles of 45 sec at 94 deg C., 60 sec at 55 deg C., 90 sec at 70 deg C.,extension at 70 deg C. for 10 min. Second round PCR conditions were: 10min initial denature at 94 deg C. followed by 35 cycles of 45 sec at 94deg C., 60 sec at 55 deg C., 60 sec at 70 deg C., extension at 70 deg C.for 10 min. DNA fragments were purified with Quickclean II PCR Kit(GenScript), cloned into pDrive (Qiagen) and sequenced.

PfSEP-1 Knock Out/Down Strategy

We constructed vectors designed to disrupt the promoter region(knockdown) and the coding region (knock-out) of the gene encodingPfSEP-1. For the knock-down construct, we amplified a 749 bp segment(−493-257 bp) from 3D7 genomic DNA using PCR forward primers5′-GCACTGCAGAGCACTGAATAAATGAAATG-3′(SEQ ID NO: 53) and reverse primer5′-GCAGCGGCCGCGTGGATGCACCATCATCGAG-3′ (SEQ ID NO: 54). For the knockoutconstruct, we amplified a 868 bp segment (232-1099 bp) from 3D7 genomicDNA using PCR forward primers 5′-GCACTGCAGGAGTTATCTCGATGATGGTG-3′ (SEQID NO: 55) and reverse primer5′-GCAGCGGCCGCGATCCATGATATTAACATGGCTC-3′(SEQ ID NO: 56).

Amplified DNA fragments were digested with the restriction enzymes PstIand NotI and cloned into plasmid pHD22Y 30. The DNA sequences andlocation of all inserts were confirmed by using vector specific primersin the sequencing reaction which spanned the cloning region of thevector.

Asexual stages of W2 and 3D7 parasites were cultured as described above.The parasites were synchronized using 5% d-sorbitol, and schizont stagesat 10% parasitemia were purified using a Percoll-sorbitol separationmethod 31. Uninfected RBCs were electroporated with 200 lag ofsupercoiled pHD22Y containing DNA inserts as described 9′32. Followingtransformation, purified schizonts were added to electroporated RBCs andwere maintained in culture for 48 h before the addition of drug WR99210(Sigma) to a final concentration of 5 nmole/L. Drug-resistant parasitesappeared three to four weeks after transfection. Episomal carriage ofplasmids in the drug resistant parasites was confirmed by PCR for bothconstructs using genomic DNA obtained from the drug resistant parasitesand vector specific primers F 1 5′-CATGTTTTGTAATTTATGGGATAGCG-3′(SEQ IDNO: 57) and R1 5′-CGCCAAGCTCGAAATTAACCCTCAC-3′(SEQ ID NO: 58). Six toeight weeks after transfection, we tested for chromosomal integrationfor both constructs by PCR using genomic DNA obtained from the drugresistant parasites and chromosomal and vector specific primers F25′-GCCACATATAATTCTTGTACTTGTC-3′ (SEQ ID NO: 59) and R25′-CGAAATTAACCCTCACTAAAGG-3′ (SEQ ID NO: 60) or R35′-GACAAGTACAAGAATTATATGTGGC-3′ (SEQ ID NO: 61) for knockdownconstructs, or F2 5′-GTATGATGGAAAATAAATACCCAAATG-3′(SEQ ID NO: 62) andR2 CGAAATTAACCCTCACTAAAGG-3′ (SEQ ID NO: 63) or R35′-GACAAGTACAAGAATTATATGTGGC-3′(SEQ ID NO: 64) for knockout constructs(FIGS. 16A-C).

Anti-PfSEP-1 Antibody Assays

Initial, confirmatory antibody assays were performed with rPfSEP-1Acoated ELISA plates according to known methods (FIG. 18).

To measure IgG anti-rPfSEP-1A antibody levels in the entire cohort, abead-based assay was used. 100 μg of rPfSEP-1A or 100 ug of BSA wasconjugated to 1.25×10⁷ microspheres (Luminex) and conjugated rPfSEP-1and BSA beads were pooled and lyophilized in single use aliquots.Reconstituted beads were incubated for 30 min at 37 deg C. with humanplasma samples at 1:80 dilution in Assay Buffer E (ABE, PBS pH 7.4containing 0.1% BSA, 0.05% Tween-20, and 0.05% sodium azide) inmicrotiter filter bottom plates (Millipore). Beads were washed threetimes in ABE by vacuum filtration and incubated for 30 min at 37 deg C.with biotinylated antihuman IgG (Pharmingen) diluted 1:1000 in ABE.Beads were washed three times in ABE by vacuum filtration and incubatedfor 10 min at 37 deg C. with phycoerythrin conjugated streptavidin(Pharmingen) diluted 1:500 in ABE. Beads were washed three times in ABEby vacuum filtration, resuspended in ABE and analyzed on a BioPlex 200multi-analyte analyzer. Fluorescence values for BSA beads weresubtracted from rPfSEP-1A beads. The cut-off for detectable anti¬PfSEP-1antibody levels was defined as fluorescence values greater than themean+2SD fluorescence level of 95 healthy North American children.

Growth Inhibition Assays

Growth inhibition assays (GIA) were carried out with anti-PfSEP-1 mousesera or controls. Sera were dialyzed overnight in PBS, pH7.4, heatinactivated at 56° C. for 30 min and pre-incubated with human RBC for 1hour before use in GIA assays. GIA assays were carried out using W2, 3D7and D10 strains of P. falciparum. Parasites were synchronized to thering stage by treatment with 5% sorbitol 34 for three successivereplication cycles and cultured to the mature trophozoite stage.Parasites at 0.3-0.4% parasitemia and 2% hematocrit were incubated withanti-sera at a final concentration of 10% in a final volume of 100 μl inmicrotiter wells. Cultures were performed in triplicate with fivereplicates (comprising a total of 15 individual wells) prepared for eachtreatment condition. After 24 hr, blood films were prepared from eachreplicate, stained with Giemsa, ring stage parasites were enumerated,and the results from the three wells were averaged.

Schizont Arrest Assays

Schizont arrest assay (SAA) were carried out with anti-PfSEP-1 mousesera or controls. Sera were dialyzed overnight in PBS, pH7.4, heatinactivated at 56° C. for 30 min and pre-incubated with human RBC for 1hour before use in SAA assays. SAA assays were carried out using W2 and3D7 strains of P. falciparum. Parasites were synchronized to the ringstage by treatment with 5% sorbitol 34 for three successive replicationcycles and cultured to the early-schizont stage. Parasites at 3.5%parasitemia and 2% hematocrit, consisting mainly of early schizonts wereincubated with anti-sera at a final concentration of 10% in a finalvolume of 100 pl in microtiter wells. Cultures were performed intriplicate with five replicates (comprising a total of 15 individualwells) prepared for each treatment condition. After 12 hr, blood filmswere prepared from each replicate, stained with Giemsa, schizont stageparasites were enumerated, and the results from the three wells wereaveraged.

Immunofluorescence Assays

Blood smears of asynchronous 3D7 strain parasite cultures were prepared,fixed in cold methanol for 15 minutes, and probed with anti-PfSEP-1prepared by DNA vaccination, pre-immune sera, or rabbit anti-PfMSP-1(MR4) diluted 1:200 in PBS, 5% BSA, pH 7.4. Blood smears were incubatedwith primary antibodies for 1 hr at 25 deg C., washed three times inPBS, 0.05% Tween-20 and incubated with goat anti-mouse IgG conjugatedwith Alexa fluor 488 (Molecular Probes) and goat anti-rabbit IgGconjugated with Alexa Fluor 594 (Molecular Probes). Blood smears wereincubated for 10 minute in 1 lig/ml of 4′,6′-diamino-2-phenylindole(DAPI, Sigma) to label nuclei and cover slipped with ProLong Goldanti-fade reagent (Invitrogen). Blood smears were imaged using aconfocal microscope (Leica SP2, Leica Microsystems, Exton, Pa.) equippedwith a 100× oil immersion objective and sequential Z-sections of theinfected RBC were collected.

For localization of PfSEP-1 in late stage schizonts, we performed livecell staining and imaging. Briefly, 3D7 strain parasites weresynchronized to the ring stage by treatment with 5% sorbitol 34 forthree successive replication cycles and cultured to the early-schizontstage. Anti-PfSEP-1 prepared by DNA vaccination (1:200) and rabbitanti-human glycophorin A (1:200) were incubated with live schizontinfected RBCs in PBS, 5% BSA pH 7.4 for one hr at 25 deg C. Samples werewashed three times in PBS and incubated with goat anti-mouse IgGconjugated with Alexa Fluor 594 (Molecular Probes) and goat anti-rabbitIgG conjugated with Alexa Fluor 488 (Molecular Probes). Samples werewashed 3 times with PBS and incubated for 10 minute in 1 μg/ml of4′,6′-diamino-2-phenylindole (DAPI, Sigma) to label nuclei. Blood smearswere prepared and cover slipped with ProLong Gold anti-fade reagent(Invitrogen). Blood smears were imaged using a confocal microscope(Leica SP2, Leica Microsystems, Exton, Pa.) equipped with a 100× oilimmersion objective and sequential Z-sections of the infected RBC werecollected.

Immunoelectron Microscopy

3D7 strain parasites were synchronized to the ring stage by treatmentwith 5% sorbitol 34 for three successive replication cycles and culturedto the early-schizont stage. Samples were blocked for 1 hour at 25 degC. in I×PBS containing 2% BSA. Samples were incubated with anti-PfSEP-1prepared by DNA vaccination (diluted 1:50 in PBS) and rabbit anti-humanglycophorin-A polyclonal sera (diluted 1:50 in PBS) for 3 hr at 25 degC. Pre-immune mouse sera was used as a negative control. Samples werewashed three times in 1×PBS, and incubated for 1 h at 25 deg C. with 5or 18-nm gold-conjugated goat anti-mouse IgG (Invitrogen) and 10-nmgold-conjugated goat anti-rabbit IgG (Invitrogen). Samples were washedthree times in I×PBS, and were fixed for 30 min at 4° C. with 2%glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium cacodyldate buffer.Samples were dehydrated, embedded in Epon (EMS), sectioned on anultra-microtome, counter stained for 10 min in 5% aqueous uranyl acetateand examined on a Philips CM10 electron microscope.

PbSEP-1A Antibody and Vaccination Studies

Antibody assays were performed with rPbSEP-1A coated ELISA platesaccording to our published methods 14 using an HRP conjugated anti-MouseIgG antibody (Sigma) for detection of bound anti-PbSEP-1A antibodies.

We immunized Balb/C mice (n=11) with 40 ug of rPbSEP-1A emulsified in100 ul of TiterMax Gold adjuvant or adjuvant alone (n=11). Mice wereimmunized IP on days 0, 14, 28, and 42 and SC on day 56. On day 63, micewere challenged IP with 106 P. berghei ANKA parasite infected red bloodcells. Mice were monitored daily from day 4 post-challenge with bloodfilms to quantify parasitemia. Mice with parasitemias greater than 20%or exhibiting signs of illness (hunching, immobility, decreased foodintake, etc.) were euthanized.

Statistical Analyses

To assess the relationship between anti-PfSEP-1 antibody responses andresistance to clinical malaria outcomes, we developed repeated measuresmodels using SAS version 9.3 (Cary, N.C.). Generalized estimatingequations using quasi-likelihood estimation were employed for thesecorrelated (repeated measures) binary outcome data (Zeger, S. L. &Liang, K. Y. Longitudinal data analysis for discrete and continuousoutcomes. Biometrics 42, 121-130 (1986)). Proc Genmod with a binomialdistribution and logit link function were specified with separate modelsfor each of the dichotomous clinical malaria outcomes. Due to the lackof independence of the repeated measures on children over time, weutilized longitudinal (repeated measures) modeling techniques in ProcGenmod to adjust for the correlation of responses within individuals. Anautoregressive correlation structure was chosen given the expectationthat the correlation of responses will decline over time. The fit of themodel with different correlation structures was evaluated with theQuasi-Akaike Information Criterion (QIC). Similar GEE based linearregression models were used for the continuous endpoints of parasitedensity on all available blood smears and parasite density on positiveblood smears. For some dichotomous malaria outcomes, including severemalaria, sampling zeros (i.e. no cases of severe malaria) occurred amongchildren with detectable anti-PfSEP-1 antibody responses. This leads to“infinite bias” whereby odds ratios are skewed far above the true oddsratio. To address this, we used the Laplace correction, adding oneadverse event to the group with detectable anti-PfSEP-1 antibody levelsand a proportional number of events to the group with undetectableanti-PfSEP-1 antibody levels to restore the discordant pair ratios(Greenland, S., Schwartzbaum, J. A. & Finkle, W. D. Problems due tosmall samples and sparse data in conditional logistic regressionanalysis. Am J Epidemiol 151, 531-539 (2000)).

The data from these studies indicate that resistant individuals had 4fold higher antibody levels to recombinant Pf SEP-1 compared tosusceptible individuals, anti-Pf SEP-1 detects a 244 kDa antigen in P.falciparum infected, but not uninfected RBCs, Pf SEP-1 localizes to theschizont/parasitophorous vacuole membrane, Mauer's clefts and the innerleaflet of the RBC membrane in schizont infected RBCs, anti-Pf SEP-1inhibits parasite growth by 48-74%. In schizont arrest assays, anti-PfSEP-1 inhibits schizont rupture by 4-7 fold, and PfSEP-1 is a usefulvaccine antigen to target schizont rupture and thereby reduce theseverity of malaria.

Example 2: Role of Phosphorylation and Protein-Protein Interaction inSchizont Egress

PfSEP-1 is involved in the process of schizont egress from P. falciparuminfected RBCs. As was described above, PfSEP-1, a 244-kDa parasiteantigen, localizes to the schizont/parasitophorous vacuole membrane,Maurer's clefts and the inner leaflet of the RBC membrane in schizontinfected RBCs. Antibodies to a central, highly conserved 274 aa regionof PfSEP-1 (rPfSEP-1A, aa 810-1083) decrease parasite replication by58-75% (all p<0.009) by blocking schizont rupture. Active vaccinationwith rPbSEP-1A results in a 2.25 fold reduction in parasitemia after invivo challenge with P. berghei. In human cohort studies, childrenexperienced a dramatically increased incidence of severe malaria duringperiods with undetectable anti-PfSEP-1 antibody levels (45 cases/23,806child weeks) compared to periods with detectable antibody levels (0cases/1,688 child weeks; adjusted OR 4.4; Type III fixed effectsp<0.01). These results demonstrate that PfSEP-1 is critical for parasiteegress and that antibodies against this protein are protective in vivoagainst severe malaria.

Schizont egress is a complex and tightly regulated process that requiresboth calcium-signaling and activation of a protease cascade whichprocesses both parasite and host RBC proteins. Central events includeactivation of PfPKG, release of PfSUB1 into the parasitophorous vacuole,and proteolytic processing/activation of PfSERA5 by PfSUB1. Conditionalknockdown of the calcium dependent kinase PfCDPK5 also results in arrestof schizont egress. Vaccination with PfSERA5 reduces and blocks schizontegress as well as parasite invasion. An in vivo phosphorylationsubstrate(s) of PfCDPK-5 is PfSEP-1.

Protein-protein interactions of PfSEP-1 were studied using yeasttwo-hybrid (Y2H) and focusing on the rPfSEP-1A region (aa 810-1083; SEQID NO:2) and confirmed by immunoprecipitation of schizont extracts withanti-PfSEP-1 and sequencing (FIG. 20). PfSEP-1 was cloned into a “bait”plasmid as fusion with truncated transcription factor; malaria cDNAswere cloned into target plasmid as fusion with truncated transcriptionfactor; screening was carried out in yeast for complementation oftranscription factor via reporter gene assay; and PfSERA5 was identifiedas binding partner for PfSEP-1. The analysis also identified PfMESA asbinding partner. These screens have identified 26 potential interactingproteins including PfSERA5, PfEMP2 (MESA), RAP-1, and RhopH3, which havealso been identified as substrates for the egress critical proteasePfSUB1. An immune response against SERA5 and SUB 1 sequences inhibitschizont egresss. SERA5 was identified in yeast-2-hybrid screen usingPfSEP-1A as bait. rPfCDPK-5 was found to phosphorylate rPfSEP-1A (seeFIGS. 20-21).

Phosphorylation-mediated regulation of PfSEP-1 and binding of thisprotein to both parasite and RBC proteins is essential for parasiteegress. Parasite and RBC proteins which interact with, or phosphorylatePfSEP-1, are useful as vaccine antigens alone or together with PfSEP-1(e.g., PfSEP-1A peptide) for immunization against malaria. Thus,plasmodial kinases (e.g., Pf CDPK5) and PfSEP-1-interacting proteins(e.g., PfSERA5, PfEMP2 (MESA), RAP-1, RhopH3) are used alone or ascomponents of an PfSEP-1 based vaccine composition to generate anantibody or cellular immune response, which leads to a synergisticreduction in parasite growth, schizont egress, and (as a result)reduction in severity of malaria.

Example 3: Transmission Blocking and Reduction of Mosquito Invasion

Gametocytes, a form of blood stage parasite, are picked up by a femaleAnopheles mosquito during a blood meal. PfSEP-1 is expressed in male andfemale gametocytes—the sexual stage of the parasite's development thatforms within host red blood cells. After being taken up by the mosquitowith a blood meal, gametocytes must rupture from their encasing redblood cell in a process analogous to schizont rupture. This processtakes place within the gut of the mosquito. Male and female gametocytesthat fail to rupture from their red blood cell cannot join to make anookinete and thus cannot infect the mosquito.

Several transmission blocking vaccine candidates attempt to targetookinete development in the mosquito (Kaslow et al., Infect Immun 1994;62:5576-80; Bustamante et al., Parasite Immunol 2000; 22:373-80).Because PfSEP-1 is expressed in gametocytes (FIGS. 18 E-G), antibodiesto PfSEP-1 taken up with the blood meal prevent gametocyte rupture fromhost red blood cells within the mosquito, thus affording a transmissionblocking effect. Thus a vaccine that elicits an antibody immune responseagainst PfSEP-1 (e.g., antibodies that specifically bind to PfSEP-1A)also leads to blocking of gametocyte egress out of RBCs. Antibodies madeas a result of the vaccination regimen described herein readily gainaccess to the RBC, because the membrane permeability of infected RBCs.Thus, these data indicate that the vaccine is also useful to prevent orreduce invasion of mosquitos from a human blood meal.

Example 4: Vaccination of Mothers and Adolescents

Maternal transmission of anti-PfSEP-1 antibodies from a mother to afetus, e.g., across the maternal-fetal interface via the placenta, wasfound to reduce malaria in infants. We have identified PfSEP-1antibodies in the sera of pregnant women whose children were protectedfrom severe malaria during infancy (first yr of life), but do not detectanti-PfSEP-1 antibodies in pregnant women whose children do have severemalaria during infancy. Because neonates (first 28 days of life) havepoorly developed immune systems, they often do not make robust immuneresponses to vaccines. The vaccine described herein is therefore alsouseful to protect infants. Pregnant women and/or women of child bearingage are immunized with a vaccine containing PfSEP-1 peptide(s).Anti-PfSEP-1 antibodies produced as a result of the immunization crossthe placenta and protect the newborn from malarial infection, morbidityand mortality. Females are immunized starting at age 9, e.g., 3 dosesover 6 months. Immunization of females prior to pregnancy or early inpregnancy is useful to prevent, slow, or inhibit infection and thedevelopment of malaria in fetuses and newborns.

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank, NCBI, and Plasmodb submissionsindicated by accession number cited herein are hereby incorporated byreference. Plasmdb.org sequence version is the version as of Nov. 30,2012. All other published references, documents, manuscripts andscientific literature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A vaccine for reducing the severity of malaria or immunizing againstmalaria comprising a composition that leads to inhibition of parasiteegress from red blood cells or inhibits parasite egress from said redblood cells, wherein said composition comprises a purified PfGARPpolypeptide antigen comprising the amino acid sequence of PfGARP (SEQ IDNO:27) or a fragment thereof, said fragment thereof comprising the aminoacid sequence of PfGARP (SEQ ID NO:26), further comprising an adjuvant.2. The vaccine of claim 1, wherein said composition comprises a purifiedpolypeptide comprising the amino acid sequence of SEQ ID NO:26.
 3. Thevaccine of claim 1, wherein said composition further comprises apurified polypeptide comprising the amino acid sequence of CDPK5 (SEQ IDNO:47), SERA5 (SEQ ID NO:70, 72), PfSUB1 (SEQ ID NO:74), or PfPKG (SEQID NO:76).
 4. The vaccine of claim 1, wherein said composition comprisesa purified polypeptide that elicits an antibody immune response againstPfGARP.
 5. The vaccine of claim 1, wherein said composition comprises apurified anti-PfGARP antibody or antigen binding fragment thereof. 6.The vaccine of claim 1, wherein said antibody binds to an antigencomprising a polypeptide having at least 70% identity with an amino acidsequence of SEQ ID NO: 26 or 27 or a fragment thereof.
 7. The vaccine ofclaim 1, wherein said composition further comprises a purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, 14, 18, 22, 30, 34, 38, 42, 46, 66 and 72.8. The vaccine of claim 1, wherein said composition further comprises apurified polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 8, 11, 15, 19, 22, 31, 35, 39, 43, 47,67, 70, 74, and 76 or fragments thereof.
 9. The vaccine of claim 1,wherein said composition further comprises a purified polypeptideconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 30, 31, 34, 35, 38,39, 42, 43, 46, 47, 66, 67, 70, 72, 74, and 76 or an immunogenicfragment thereof.
 10. The vaccine of claim 1, wherein said compositionfurther comprises a purified polypeptide comprising an amino acidsequence of 6, 7, 10, 11, 14, 15, 18, 19, 22, 23, 30, 31, 34, 35, 38,39, 42, 43, 46, 47, 66, 67, 70, 72, 74, and/or
 76. 11. The vaccine ofclaim 1, wherein said antigen comprising a polypeptide encoded by apolynucleotide having at least 70% identity with the nucleic acidsequence of SEQ ID NO:
 25. 12. The vaccine of claim 11, wherein saidpolypeptide encoded by a polynucleotide having the nucleic acid sequenceof SEQ ID NO:
 25. 13. An isolated peptide comprising a peptide having atleast 95% to 99% identity with the sequence of SEQ ID NO: 27 or afragment thereof, wherein said peptide or fragment thereof elicits ananti-PfGARP antibody response against P. falciparum malaria, furthercomprising an adjuvant.
 14. The peptide of claim 13, wherein saidpeptide comprises SEQ ID NO: 27 or a fragment thereof, wherein saidfragment comprises the amino acid sequence of SEQ ID NO:26.
 15. Apurified antibody that specifically binds to an antigen comprising apolypeptide having at least 70% identity with an amino acid sequence ofSEQ ID NO: 26 or 27 or a fragment thereof.
 16. A method of treating orreducing the severity of P. falciparum malaria in a subject in needthereof comprising administering to said subject a compositioncomprising said anti-PfGARP antibody of claim
 15. 17. The method ofclaim 16, wherein said subject is a mammal.
 18. The method of claim 16,wherein said subject is a human.
 19. The method of claim 18, whereinsaid subject is at least about 6-8 weeks of age.
 20. The method of claim18, wherein said subject is an adolescent female or a female ofchildbearing age.
 21. The method of claim 16, wherein said compositionis administered intramuscularly.
 22. The method of claim 16, furthercomprising administering said composition to said subject in combinationwith a secondary therapy or a secondary agent.
 23. The method of claim16, said second agent comprises RTS,S (Mosquirix) or MSP-1.